HOROWITZ 

Study  of  the  Action  of  Ammonia 
on  Thymol 


QD 
341 
P5 
H25 


A  Study  of  the  Action  of  Ammonia 
on  Thymol 


DISSERTATION 

SUBMITTED    IN    PARTIAL    FULFILMENT    OF    THE    REQUIRE- 
MENTS FOR.  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 
IN  THE  FACULTY   OF  PURE  SCIENCE   OF 
COLUMBIA   UNIVERSITY 


BY 

BENJAMIN  HOROWITZ,  CHEM.,  M.  A. 

NEW  YORK   CITY 
1913 


NEW  YORK 

SCHOEN  PRINTING  COMPANY 
1913 


)  A  Study  of  the  Action  of  Ammonia 
on  Thymol 


DISSERTATION 

SUBMITTED    IN    PARTIAL    FULFILMENT    OF    THE    REQUIRE- 
MENTS FOR  THE  DEGREE  OF  DOCTOR   OF  PHILOSOPHY 
IN   THE   FACULTY   OF  PURE  SCIENCE   OF 
COLUMBIA   UNIVERSITY 


BY 

BENJAMIN  HOROWITZ,  CHEM.,  M.  A. 

NEW  YORK   CITY 
1913 


NEW  YORK 

SCHOEN  PRINTING  COMPANY 
191} 


To  My  Mother  and  Father 


ACKNOWLEDGMENT. 


The  author  wishes  to  take  this  opportunity  of  expressing  his 
indebtedness  to  Professor  William  J.  Gies,  who  suggested  the  in- 
vestigation, and  under  whose  direction  it  was  carried  out. 

Prof.  Gies'  encouragement,  constant  good  counsel,  and  his 
many  acts  of  kindness,  are  gratefully  appreciated  by  the  author. 

B.  H. 

Laboratory  of  Biological  Chemistry, 
College  of  Physicians  and  Surgeons, 
Columbia  University,  May,  1913. 


TABLE  OF  CONTENTS. 


PAGE 

Dedicatory  2 

Acknowledgment  3 

Introductory  5 

History  of  Thymol  7 

The  Action  of  Ammonia  on  Phenols  Other  Than  Thymol 23 

The  Action  of  Ammonia  on  Thymol 28 

Is  the  Blue  Color  due  to  an  Impurity  ? 30 

Certain  Quantitative  Relationships  between  Thymol,  Ammonia  and 

Alcohol,  and  Thymol  and  Ammonia  Alone 33 

Accelerating,  Indifferent,  and  Retarding  Agents  in  the  Formation  of 

the  Blue  Color  38 

Are  Ammonia  and  Thymol  Alone  the  Active  Factors  in  the  Formation 

of  the  Blue  Color? 40 

Effect  of  Several  Reagents  upon  the  Blue  Solution 46 

Tests  for  the  Presence  of  Nitrogen  in  the  Pigment 47 

Isolation  of  the  Blue  Product 51 

The  Chemical  Constitution  of  the  Pigment 55 

The  Action  of  Sodium  Hydroxide,  Potassium  Hydroxide,  and  Barium 

Hydroxide  on  Thymol  57 

Addendum  62 

Summary  of  General  Conclusions  63 

Bibliography  65 

Biographical  67 

Publication  ..  ..68 


INTRODUCTION. 


In  the  course  of  experiments  on  enzymes  as  possible  factors 
in  the  development  of  edema  Prof.  Gies,  greatly  to  his  surprise, 
found  that  trypsin  in  ammonium  hydroxide  solution  containing 
sodium  chloride,  failed  to  give  the  swelling  results  with  elastin 
which  had  previously  been  observed  under  similar  conditions; 
instead  of  swelling,  the  elastin  particles  gradually  became  green 
and  then  blue  (1).  It  was  then  recalled  that  the  elastin  used  had 
been  prepared  some  years  before,  and  had  been  preserved  with  a 
solution  of  thymol  in  alcohol.  That  the  blue  color  was  due  to 
thymol  was  confirmed  by  mixing  some  thymol  with  ten  per  cent, 
ammonium  hydroxide  solution  and  obtaining  the  color  on  stand- 
ing. Alcohol  appeared  to  accelerate  the  transformation.  By 
evaporating  the  ether  extract  (which  was  red),  a  purplish  red 
oily  product,  soluble  in  ether,  toluene,  and  alcohol,  was  obtained. 
On  another  occasion  the  oily  product  became  crystalline. 

One  of  the  most  interesting  observations  was  that  "the  red 
alcoholic  solution  was  turned  deeply  bluish  by  a  drop  of  N/10 
sodium  hydroxide  solution,  the  red  being  restored  by  a  drop  of 
N/10  hydrochloric  acid  solution."  This  color  reaction  would 
clearly  indicate  its  use  as  an  indicator. 

These  observations  naturally  suggested  further  inquiry. 
What  was  the  nature  of  the  blue  pigment  obtained  by  the  action 
of  ammonia  upon  thymol?  How  would  it  behave  when  substi- 
tuted for  thymol  in  the  important  uses  to  which  the  latter  is 
put?  How  would  other  phenols  behave  when  treated  with  am- 
monia, and  how  would  the  thymol  pigment,  and  the  other  phenol 
derivatives  compare  chemically  and  pharmacologically? 

It  was  with  this  broad  object  in  view  that  the  present  inves- 
tigation was  undertaken. 

Here  the  more  purely  chemical  side  of  the  problem  has 
been  dealt  with,  for  within  the  given  time  little  more  could  be 
done;  but  pharmacological  studies  have  already  been  begun,  and 
investigation  of  various  other  aspects  of  the  subjects  are  under 
way. 


HISTORY  OF  THYMOL 

Thymol,  a  derivative  of  cymene,  and  hence  belonging  to 
the  important  class  of  organic  compounds  known  as  the 
terpenes  and  camphors,  has  engaged  the  attention  of  the 
chemist  for  many  years.  Its  extensive  use  in  medicine — 
the  latest  application  in  hookworm  disease  (2)  being  worthy 
of  special  mention — has  increased  the  necessity  for  further 
exact  study.  L,enher's  use  of  it  in  a  colorimetric  deter- 
mination of  titanium  (3)  tends  to  show  that  it  is  also  of  value 
to  the  analytical  chemist. 

EARLY   HISTORY 

Prior  to  1853,  thymol  was  known  under  such  names  as 
"thymolic  acid,"  "thyme  oil,"  "stearoptene,"  "thymol 
hydrate,"  and  "thymene  oil  camphor."  Just  who  discov- 
ered it,  or  who  first  extracted  it  from  one  of  the  several  vola- 
tile oils  in  which  it  occurs,  is  not  known.  There  is  good 
reason  to  believe  that  the  Indians  were  familiar  with  it  long 
before  it  was  known  in  Europe(4,  5).  In  1719  Kaspar 
Neumann,  court  apothecary  in  Berlin,  observed  that  crys- 
tals were  deposited  from  the  oil  of  thyme (6).  Brown (6,  7) 
stated  that  this  substance  had  long  been  known  in  England 
under  the  name  of  Sal  volatile  thymi,  but  that  it  was  quite 
different  from  camphor.  Not  till  1846  was  anything  definite 
known  with  regard  to  it.  In  that  year  Arppe(8)  published 
an  excellent  paper  on  the  volatile  oil  of  Monarda  punctata 
("Horse  mint"),  wherein  he  showed  that  the  oil  consisted 
essentially  of  two  substances,  an  "elaeoptene"  and  a  "stea- 
roptene." The  latter,  which  we  now  know  to  be  thymol, 


was  further  purified  by  steam  distillation,  and  an  analysis 
yielded  the  formula  C10H14O— the  one  accepted  to-day. 
Arppe  found  its  melting  point  to  be  48°,  and  noticed  that 
when  heated  beyond  this  point,  the  "stearoptene"  remained 
in  a  fluid  condition  even  after  room  temperature  had  been 
reached.  The  insertion  of  a  crystal  of  the  substance  caused 
the  solidification  of  the  whole  mass. 

Doveri(9),  in  an  investigation  of  the  oil  of  thyme,  found 
that  upon  standing  it  deposited  a  "stearoptene."1  The  oil, 
which  at  first  was  reddish  brown  in  color,  could  be  converted 
to  a  light  yellow  by  repeated  distillation.  The  portion, 
which  boiled  at  a  temperature  between  230-235°,  gave 
results,  upon  analysis,  that  indicated  a  composition  corre- 
sponding to  the  formula,  C10H15O. 

Relationship  to  Phenol 

Historically,  the  most  important  contribution  to  our  knowl- 
,edge  of  thymol  is  undoubtedly  Lallemand's  who,  in  a  brilliant 
series  of  researches (10)  extending  between  1853-1857,  clearly 
showed  its  relationship  to  phenol.  He  first  found  that  the 
oil  of  thyme  consists  of  a  substance  containing  oxygen, 
C10HUO  (which  he  called  "thymol"),  and  of  a  hydrocarbon, 
C10H16.  The  thymol  could  be  readily  extracted  with  alkalis, 
and  reprecipitated  by  the  addition  of  hydrochloric  acid. 
Its  melting  point  was  recorded  as  44°,  and  the  boiling  point 
230°.  Sulphuric  acid  was  found  to  dissolve  it  quite  readily, 
and  the  resulting  substance,  obtained  by  the  evaporation 
of  the  solution,  was  soluble  in  water. 

In  his  next  paper  Lallemand  deals  with  the  action  of 
oxidizing  agents.  By  mixing  thymol  with  H2SO4  and  MnO2 
and  distilling,  orange-yellow,  oily  drops  were  obtained  which 
soon  solidified.  This  substance,  known  to  us  as  thymo- 
quinone,  and  which  Lallemand  called  "thymol,"  was  found 
to  be  analogous  to  quinine.  This  "thymol"  could  be  re- 

1  Many  oils  which  can  be  distilled  either  alone  or  with  steam,  without 
undergoing  decomposition — the  so-called  volatile  or  essential  oils — deposit 
a  solid  (stearoptene)  on  cooling,  leaving  a  liquid  portion  (elaeoptene) . 


duced  with  sulphur  dioxide,  giving  "thymoilol"  (thymo- 
hydroquinone),  analogous  to  hydroquinone.  To  complete 
the  analogy,  "thymoil"  and  "thymoilol"  combine  similarly 
to  quinone  and  hydroquinone. 

In  his  further  contributions  Lallemand  brought  forward 
still  more  evidence  to  prove  his  contention  that  thymol  was 
a  homologue  of  phenol.  With  that  object  in  view,  he  pre- 
pared the  di-  and  tri-nitro  derivatives,  and  also  studied  the 
action  of  chlorine. 


Identical  Products  Obtained  from  Various  Volatile  Oils 

Haines(n)  investigated  the  oil  of  Ptychotis  ajowan,  ob- 
tained in  Rajpootana,  and  which  the  native  doctors  used. 
The  "  stearoptene "  obtained  from  it  was  found  to  be  iden- 
tical with  the  crystals  sold  in  the  bazaar  under  the  name 
of  Ajwa  Ka  Phul  (flowers  of  Ajwa).  "  I  have  not,"  he  writes, 
"been  able  to  discover  by  what  method  the  natives  contrive 
to  obtain  the  stearoptene  from  the  oil:  it  is  probably  so 
loaded  with  it  as  to  crystallize  out  on  dropping  in  a  frag- 
ment ready  formed,  without  it  being  necessary  to  redistil 
the  oil."  Of  great  importance  is  his  finding  that  his  com- 
pound is  identical  with  the  one  obtained  by  Lallemand  from 
the  oil  of  thyme,  though  he  adds,  curiously  enough,  "I  could 
not  observe,  however,  the  property  which  Lallemand  as- 
signs to  thymol,  of  combining  with  caustic  alkalis." 

Stenhouse(i2)  came  to  the  same  conclusion  as  Haines, 
and  found  in  addition  that  his  stearoptene  from  Ptychotis 
ajowan  agreed  in  every  way  with  Arppe's  from  monarda 
oil  and  Doveri's  from  the  oil  of  thyme. 

Most  striking  was  Kolbe  and  Lauterman's  work (13)  in 
showing  how  close  was  the  relationship  existing  between 
phenol  and  thymol.  These  workers,  whose  researches  upon 
the  action  of  phenols  in  the  presence  of  sodium  and  carbon 
dioxide  have  become  classical,  showed  that  thymol  could  be 
converted  to  thymotic  acid  in  the  same  way  as  phenol  could 
be  transformed  into  salicylic  acid. 


Effect  of  Kekule's  Benzene  Theory 

Kekule"'s  benzene  theory,  advanced  in  1865,  had  no  small 
influence  upon  the  views  taken  with  regard  to  the  consti- 
tution of  thymol  (7),  as  indeed  it  had  with  regard  to  the  con- 
stitution of  phenols  in  general.  Now  for  the  first  time  a 
sharp  differentiation  came  to  be  made  between  the  aromatic 
alcohols  which,  on  oxidation,  yielded  the  corresponding  alde- 
hydes and  acids,  and  their  isomers,  the  phenols,  which  did 
not  yield  acids  containing  the  same  number  of  carbon  atoms. 
But  this  did  not  differentiate  the  phenols  from  the  tertiary 
alcohols;  and  so,  as  characteristic  of  the  former,  it  was  stated 
that  they  dissolved  in  caustic  alkalis,  the  hydrogen  of  the 
hydroxyl  being  replaced  by  sodium — a  reaction  which,  in  the 
case  of  the  alcohols,  could  be  accomplished  only  by  the  ac- 
tion of  the  alkali  metals. 


Relationship  to  Cymene  and  to  Carvacrol 

Considerable  progress  on  the  constitution  of  thymol  was 
made  by  the  researches  of  Engelhard t  and  Latschinoff(i4). 
By  fusing  cymene  sulphonic  acid  with  potassium  hydroxide 
they  obtained  a  phenol  which,  though  not  thymol,  was  found 
to  be  isomeric  with  it.  Upon  examination  they  found  it  to 
be  identical  with  a  phenol  similarly  isolated  by  Muller(i5), 
and  which  he  regarded  as  thymol,  and  another  by  Pott(i6), 
who  first  obtained  his  cymene  from  camphor  by  the  action 
of  phosphorus  pentasulphide,  and  then  the  phenol  by  fusing 
its  sulphonic  acid  with  potassium  hydroxide.  We  now 
know  this  substance  to  be  carvacrol,  isomeric  with  thymol. 

The  synthetic  process  having  failed,  Engelhardt  and  Latschi- 
noff  substituted  analytical  methods.  Recalling  one  of  Lalle- 
mand's  experiments,  wherein  he  obtained  a  chlorine  deriva- 
tive of  thymol,  C10H8C15O,  which,  on  warming  to  200°,  broke 
down  into  propylene  and  tetrachlorcresol,  they  treated 
thymol  with  phosphorus  pentoxide,  and  obtained  propylene 
and  cresol.  Just  which  of  the  three  cresols  this  was  could 
not  be  ascertained  with  exactness,  as  little  was  known  with 


II 

regard  to  the  position  of  the  methyl  and  hydroxyl  groups. 
Only  at  a  later  period  was  it  shown  that  the  cresol  under 
question  was  identical  with  the  meta  variety. 

Carstanjen(i7)  brilliantly  followed  up  the  work  of  Engel- 
hardt  and  Latschinoff.  He  first  tried  the  action  of  reducing 
agents  upon  thymol,  such  as  zinc  dust  and  hydriodic  acid, 
but  the  results  were  unsatisfactory.  He  next  prepared  the 
chloride  C10H13C1  by  heating  with  phosphorus  pentachloride, 
but  the  product  obtained  was  very  impure,  and  distillation 
failed  to  purify  it,  for  at  120°,  the  substance  blackened; 
and  even  under  7  mm.  pressure  the  conditions  were  no 
better.  However,  the  product  was  treated  with  sodium 
amalgam  in  a  weak  acid  solution,  and  the  resulting  hydrocar- 
bon was  purified  by  repeated  distillation  over  metallic  sodium. 
Analysis  gave  the  formula  C10H14. 

But  Carstanjen's  further  work  upon  cymene  (for  that  is 
what  his  hydrocarbon  proved  to  be)  showed  perhaps  still 
greater  originality.  He  heated  cymene  with  a  mixture 
of  potassium  dichromate  and  sulphuric  acid  and  obtained  a 
white  powder  which  dissolved  in  ammonia,  and  was  re- 
precipitated  by  hydrochloric  acid.  This  proved  to  be  tere- 
phthalic  acid,  C6H4(COOH)2,  where  the  carboxyl  groups  are 
para  to  one  another.  From  this,  then,  it  followed  that  the 
methyl  and  propyl  groups  in  cymene  (and  therefore  in 
thymol,  which  had  been  reduced  to  cymene)  must  also  be 
para  to  one  another: 


CH3  COOH 

I     '  I 


H—  C           :—  H  H—  C        \C—  H 

p    H  -*         I       ii 

H-C.       .C-H  H-C.        ,C-H 

X(V^  ^C' 

'jjf  COOH 

Cymene  Terephthalic  acid 

A  further  important  conclusion  by  Carstanjen  was  that 


thymol  could  form  but  two  isomeric  phenols,  which  at  once 
becomes  evident  from  its  relationship  to  cymene: 


C3H7 


formula  i  representing  thymol  and  2  carvacrol.  As  further 
confirmation,  the  latter  with  phosphorus  pentoxide  yields 
o-cresol  and  (propylene)  in  contradistinction  to  m-cresol 
from  thymol. 

The  question  as  to  whether  the  propyl  group  was  normal 
or  iso  was  not  settled  until  some  years  later.  In  the 
meantime,  Fittica(iS)  modified  Carstanjen's  method  of  ob- 
taining cymene  from  thymol  by  acting  directly  upon  the  lat- 
ter with  phosphorus  pentasulphide,  and  thereby  obtaining 
the  hydrocarbon.  From  hasty  observations,  based  on  in- 
conclusive observations  by  previous  workers,  Fittica  arrived 
at  the  conclusion  that  the  C3H7  in  cymene  was  normal  and 
not  iso;  hence  it  followed  that  the  C3H7  in  thymol  was  also 
normal.  This  erroneous  view  prevailed  till  1891,  when 
Widman  solved  the  problem. 


Synthesis 

The  first  synthesis  of  thymol  was  accomplished  by  Wid- 
man (19).  This  research  is  a  model  of  its  kind.  In  a  former 
contribution  (20)  the  author  showed  how  w-toluidine  could 
be  obtained  from  benzaldehyde  by  first  nitrating  the  latter, 
acting  upon  the  nitro  compound  thus  formed  with  phos- 
phorus pentachloride,  obtaining  the  w-nitrobenzaldehyde,  and 


13 

reducing  this  with  zinc  and  hydrochloric  acid  to  the  tolui- 
dine: 


/CHO  /CH 

C.H/       —  »   C,H/ 

\H  \N02 


CHO  /CHC1,  /CH3 


/ 
/ 


By  a  similar  series  of  reactions  thymol  was  built  up.     Wid- 
man's  starting  point  was  cuminol,  or  ^-propylbenzaldehyde:1 


/CHO 

C-H<C,H, 

Cuminol 

HONO, 

/CHO 
CeH3^  NO2  - 
XC3H7 
Nitrocuminol 

f  H,0 

/CHO 
:.H£-  N02 
\C3H7 

PC15 

/CHC12 
C,H3^  NO2    + 
XCSH7 

POC1. 

Nitrocymyline 
e 


trocymy 
chloride 

/CHC12        5H2 


3  2         — ,32    +  2H20  +  2HC1 

\C3H7  \C3H7 

Cymidine 


HONO  /CH3 

— >  C.H3^OH     +  N2  +  H20 

C3H7  \C3H7 

Thymol 


When  NO2  is  introduced  into  the  ring  containing  CHO, 
the  NO3  enters  m-  to  CHO : 

1  The  C3H7  in  this  compound  is  iso,  though  this  was  not  known  at  the 
time. 


The  other  two  compounds  can  therefore  be  represented 
thus: 


NH2 


The  fact  that  thymol  was  obtained  from  m-nitrocuminoi 
is  in  itself  good  evidence  for  the  belief  that  the  hydroxyl 
group  in  thymol  is  ra-  with  respect  to  the  CH3.  This  view 
receives  abundant  support  in  that  w-cresol  can  be  obtained 
from  thymol.  As  the  reactions  were  never  allowed  to  go 
above  100°,  there  is  every  reason  for  believing  that  the 
propyl  group  in  thymol  stands  in  the  same  relation  to  the 
CH3-  group  as  it  does  in  cuminol;  and  for  this  again  we  have 
evidence,  since  thymol  can  be  reduced  to  cymene. 

The  Nature  of  the  Propyl  Group 

The  position  of  the  propyl  group,  whether  normal  or  iso, 
still  remained  unsettled.  This  problem  Widman  set  him- 
self to  solve,  and  in  1891  published  his  results  in  a  paper (21) 
which  was  a  worthy  successor  to  his  former  one.  The  aim 
in  view  was  to  investigate  the  propyl  group  in  cymene; 


15 

once  this  could  be  established,  the  nature  of  the  C3H7  in  thy- 
mol would  follow.  In  an  excellent  review  of  the  work  hitherto 
done  in  this  direction,  Widman  recalls  the  labors  of  such 
masters  as  Dumas,  Gerhardt,  Fittig,  Konig,  Beilstein,  Jacob- 
sen,  Robert  Meyer,  and  Kekule,  and  finds  a  total  disagree- 
ment among  them. 

The  question  at  issue  was  whether  the  cymene  was 
/>-methylpropylbenzene  or  />-methylisopropylbenzene.  Wid- 
man therefore  decided  to  prepare  synthetically  both  those 
compounds,  and  to  compare  the  products  so  obtained  with 
cymene.  He  prepared  the  normal  variety  by  the  action  of 
/>-bromtoluol  on  propyl  bromide  in  the  presence  of  sodium 
(Fittig's  synthesis) : 


CHS 


CH2.CH2.CH3. 


The  iso  modification  was  obtained  by  first  preparing 
isopropylbenzene  by  the  action  of  isopropylbromide  and  ben- 
zene in  the  presence  of  aluminium  chloride  (Friedel  and 
Craft's  synthesis), 


CH3.CH.CH, 


CH3  CH, 


i6 

then  brominating   the  product,   and  finally  treating  it  with 
methyl  bromide  in  the  presence  of  sodium: 


Br 


CH 
X\ 
CH3  CHS 


"Na"  Na"! 

Br  Br  j  CH,         CH, 


By  comparing  these  two  products  with  cymene  Widman 
came  to  the  conclusion  that  the  latter  was  methyliyopropyl- 
benzene.  A  tabulation  of  his  results  .as  he  gives  them  is 
appended : 


i7 

WWa       2      W 

A   ?  &        «»,         ? 

£>  a      s        R 


w 


>,$ p 


oP      . 


>' 

£LW 

H-    p 

fi 


. 


IS 


Several  Syntheses 

After  Widman's  observations  quite  a  number  of  chemists 
succeeded  in  synthetically  preparing  thymol  in  several  in- 
teresting ways,  one  or  two  of  these  having  since  been  made 
the  basis  for  commerical  production.  A  few  of  these  methods 
are  here  discussed. 

Beckmann  and  Eickelberg(22)  obtained  thymol  from 
menthone  by  brominating  the  latter,  and  heating  the  result- 
ing dibrommenthone  with  quinoline : 


CH, 

/CH\ 
CH2       CH2 

CH,        CO 
\CH/ 


4Br 


Meathone 


£ 

x\ 

CH2  CH2 

CH2CO 

CBr 

C,Hr 

Dibrotnmenthoae 


2HBr 


CH, 

CBr 

CH2  CH2 


Quinoline 


r 

c\ 


CH3 


CH2  CO        (— 


0 


I          II 
CH     C.OH 


u 


Ketone  form  of  thymol 


C3H7 
Thymol 


The  authors  presume  that  the  ketone  form  of  thymol  is 
an  intermediate  production,  though  they  failed  to  isolate  it. 

Dinesmann(23)  has  taken  out  a  patent  for  his  method  of 
preparing  thymol— one  that  recalls  Engelhardt  and  Latsch- 
inoff's  unsuccessful  attempts.  Having  in  mind  the  fact 
that  cymene  sulfonic  acid  when  fused  with  KOH  gives 


19 

carvacrol  and  not  thymol,  owing  to  the  sulphonic  acid  rad- 
icle being  o-  with  respect  to  the  CH3  group,  Dinesmann 
started  with  2-brom-/>-cymol, 


C3H7 


and  by  dissolving  it  in  fuming  H2SO4  (containing  15-20% 
SO3)  he  obtained  the  2-brom-5-(or  3)-sulphonic  acid: 

CH, 


(J 

S03Hl  J 

C3H7 


He  next  proceeded  to  eliminate  the  Br  by  heating  the  com- 
pound in  an  autoclave  at  170°  with  zinc  dust  and  ammonia, 
and  thereby  obtained  3-cymolsulphonic  acid  (zinc  salt), 


which  on  fusion  with  KOH  gave  thymol. 

Wallach(24)  obtained  thymol  (in  very  small  quantities  he 
states)  by  brominating  menthenone  (in  glacial  acetic  acid) 
and  warming  the  resulting  dibrommenthenone : 


20 

CH,  CH,  CH, 


CH         CO  CHBr    CO  CH 

^  CBr 

I  CH 

C  H  3    T 

Menthenone  Dibrommenthenone        Ketoae  form  of  thymo 


I 

\y^    \C-OH 

/-A 

/c 

/ 


Thymol 

a  reaction  that  shows  certain  similarities  to  Beckmann  and 
Eickelberg's. 

Semmler  and  McKenzie(25)  by  heating  buccocamphor 
with  HC1  for  two  hours  at  150-180°  obtained  a  quantitative 
yield  of  thymol: 

CH.CH<       '  CH.CH/      ' 

/\        \CH3  /\        \CH, 

CH3  CO  CH2  CO 

II  or         |         |                    — H80 

CH2  C.OH  CH2  CO                    -^ 


?  CH.CH, 

Buccocamphor  Ketone  form 


As  an  interesting  example  of  intramolecular  change  Semm- 
ler's  production  of  thymol  from  umbellulon(26)  is  worthy 
of  note: 


21 


CH, — CH 

H— C— CH3 


CH 


L 

\*,L~Lm 


Umbelluloa 


A 

CH     COH 

II 
CH 


Thymol 


23 

THE   ACTION   OF  AMMONIA   ON   PHENOLS   OTHER 
THAN  THYMOL.* 

As  far  back  as  1835  Robiquet  (27)  published  a  paper  on  the 
action  of  ammonia  upon  orcinol,  in  which  he  showed  that  am- 
monia gas  in  the  presence  of  dry  air  produced  no  color  with 
orcinol,  part  of  the  ammonia  simply  being  absorbed ;  but  as  soon 
as  moisture  was  allowed  to  enter  the  mixture,  a  blue  color  began 
to  develop.  The  resulting  product,  which  could  be  isolated  by 
extracting  the  blue  substance  with  ether  and  evaporating  the  lat- 
ter, could  not  be  obtained  in  a  crystalline  form,  and  this  fact 
prevented  Robiquet  from  pursuing  investigations  into  the  nature 
of  the  substance.  However,  he  satisfied  himself  that  it  was  not 
simply  an  ammonium  salt,  since  it  failed  to  yield  ammonia  by 
heating  with  an  alkali.  He  also  showed  that  other  alkalis  could 
not  be  substituted  for  ammonia  in  this  reaction.  Potassium 
hydroxide,  for  example,  produced  a  brown  color — one  which 
could  be  obtained  by  exposing  moist  orcinol  to  the  air.  Upon  ex- 
traction with  ether  and  evaporation  of  the  solvent,  orcinol  was 
again  obtained. 

By  a  modification  of  Robiquet's  process  for  obtaining  orcein, 
De  Luynes  (28)  succeeded  in  isolating  the  coloring  matter  of 
litmus.  Starting  with  orcinol, — which  could  be  extracted  from 
lichens, — De  Luynes  mixed  this  phenol  with  given  quantities  of 
sodium  carbonate,  water  and  ammonia,  and  heated  the  mixture 
for  4-5  days  at  60-80°.  On  diluting  and  acidifying  with  hydro- 
chloric acid  the  coloring  matter  was  precipitated. 

Bearing  in  mind  that  a  certain  analogy  existed  between  resor- 
cinol  and  orcinol,  Malm  (29)  tried  the  action  of  ammonia  upon 
the  former.  By  allowing  a  mixture  of  resorcinol,  ammonia,  and 
sodium  hydroxide  to  stand  in  a  warm  place  for  several  days,  and 
then  acidifying,  a  reddish-brown  precipitate  was  obtained,  which, 
upon  collecting  and  drying,  was  found  to  possess  a  metallic  lustre. 
The  behaviour  of  this  substance  with  acids  and  alkalis  was  found 
to  be  similar  to  that  of  litmus. 

Lex    (30)    found  that  in  the  presence  of  certain  oxidizing 

*  Ammonium  salts  are  not  considered  here.    For  a  general  discussion 
of  these  see  Hantsch:     Ber.  d.  D.  Chem.  Gesell.,  40,  3798  (1907). 


24 

agents,  such  as  bleaching  powder,  or  bromine  water, — and  even 
when  merely  exposed  to  the  air  for  a  sufficient  length  of  time, — 
a  solution  of  phenol  in  ammonia  would  turn  blue. 

With  the  object  of  employing  Lex's  reaction  as  a  test  for 
carbolic  acid,  Salkowski  (31)  made  a  more  careful  study  of  it. 
He  found  that  by  adding  ammonia  (one  part)  to  phenol  (four 
parts),  and  then  adding  a  few  drops  of  bleaching  powder  solu- 
tion (obtained  1>y  dissolving  one  part  of  bleaching  powder  in 
twenty  parts  of  water,  filtering,  and  using  the  filtrate),  and 
warming,  a  blue  color  would  instantly  make  its  appearance  if 
much  phenol  were  present.  If  the  amounts  were  small  the  color 
would  develop  in  a  few  minutes  or  in  a  quarter  of  an  hour,  de- 
pending upon  the  quantity  of  phenol.  Very  dilute  solutions  gave 
a  green  color.  Too  strong  heating,  or  the  presence  of  an  exces- 
sive quantity  of  bleaching  powder,  prevented  the  reaction. 
Salkowski  maintained  that  the  test  was  a  far  more  delicate  one 
than  with  ferric  chloride,  for  whereas  a  concentration  of 
1  : 2,000  failed  to  give  a  reaction  with  the  latter,  1  : 4,000  gave 
a  strong  blue  with  ammonia. 

In  1873  Phipson  (32)  isolated  a  product  obtained  by  the 
action  of  ammonia  on  phenol,  which  he  called  "phenolcyanine." 
He  prepared  it  by  dissolving  phenol  in  alcohol,  adding  ammonia, 
and  allowing  the  mixture  to  remain  for  some  weeks  in  a  partially 
closed  flask.  In  about  fifteen  days,  when  the  liquid  had  become 
dark  green  in  color,  twice  its  volume  of  water  and  y^  vol.  am- 
monia were  added,  and  the  mixture  again  allowed  to  stand  for 
about  six  weeks.  By  this  time  the  liquid  had  taken  a  very  tine 
blue  tint,  was  very  dark,  and  a  certain  quantity  of  phenolcyanine 
was  found  at  the  bottom  of  the  vessel  adhering  strongly  to  the 
glass.  That  which  remained  in  solution  could  be  precipitated  by 
saturating  the  liquid  with  salt.  The  product  was  collected,  dis- 
solved in  hot  alcohol  or  benzol,  and  recovered  by  evaporating  the 
solvent. 

Its  properties  were  described  as  follows :  A  dark  blue  sub- 
stance, soluble  in  alcohol,  yielding  a  fine  blue  solution,  in  ether 
reddish-blue  solution ;  in  benzol  reddish-purple  solution.  Cone, 
sulphuric  acid  gave  a  bluish-green  coloration ;  hydrochloric  acid 
had  little  action;  nitric  acid  produced  a  nitro  compound  very 


25 

different  from  picric  acid.  Slightly  soluble  in  water.  Deep  sky 
blue  by  day,  red  by  night.  Acids  reddened  the  solution,  alkalis 
bringing  back  the  blue.  Nascent  hydrogen  destroyed  the  blue 
color,  but  upon  adding  ammonia  and  exposing  to  the  air  the  color 
was  reformed. 

As  but  a  small  amount  of  substance  was  available  the 
analysis  did  not  prove  very  successful. 

In  a  succeeding  paper  (33)  he  attempted  to  show  the  rela- 
tionship between  phenylcyanine  and  indigo,  but  his  method  of 
procedure  would  have  thrown  discredit  even  upon  an  embryonic 
chemist.  Since,  he  argues,  phenolcyanine  has  twelve  carbon 
atoms,  whereas  indigo  has  sixteen,  the  problem  is  to  introduce 
four  more  carbon  atoms  into  the  former  to  convert  it  into  the 
latter.  Whereupon  he  records  with  the  greatest  gravity  how  he 
proceeded,  first,  to  melt  phenolcyanine  at  a  moderate  tempera- 
ture with  sodium  acetate,  then  to  dissolve  the  product  in  con- 
centrated sulphuric  acid,  and  finally  throw  down  the  sulpho  acid 
by  adding  an  excess  of  water.  A  similar  experiment  was  car- 
ried out  with  phenolcyanine  and  nitro-naphthalene,  using  equal 
equivalents  of  each ;  for  the  former  contained  12  carbon  atoms, 
and  the  latter  twenty;  and  12 -f- 20 -^  2  —  16,  which  gives  us 
the  number  of  carbon  atoms  in  indigo  (  !).  "These  sulpho- 
acids,"  he  writes,  "mixed  and  saturated  with  ammonia  or  am- 
monium carbonate,  gave  a  small  quantity  of  a  purple  black 
product,  insoluble  in  water  and  alcohol,  but  soluble  in  concen- 
trated sulphuric  acid,  producing  a  dark,  emerald-green  solution. 
This  product  is  very  similar,  if  not  identical,  to  the  black  indigo 
produced  when  the  leaves  are  badly  fermented." 

And  this  is  the  last  we  hear  of  Phipson ! 

A  notable  contribution  to  the  orcein  question  is  that  by 
Liebermann  (34).  In  the  course  of  a  study  of  the  action  of 
ammonia  upon  orcinol,  it  occurred  to  him  that  possibly  the  com- 
bined action  of  the  ammonia  and  the  oxygen  of  the  air  was 
equivalent  to  nitrous  acid.  Accordingly  he  dissolved  orcinol  in 
concentrated  sulphuric  acid,  and  added  potassium  nitrite,  where- 
upon a  deep  purple  coloration  was  obtained.  The  addition  of 
water  threw  down  a  red  flocculent  precipitate,  which  was  found 


26 

to  be  soluble  in  alkalis,  giving  a  beautiful  red  solution.     But  the 
substance  was  not  identical  with  orcein. 

Further  work  upon  phenols  assured  Liebermann  that  phenols 
in  general  responded  to  color  tests  with  nitrous  acid.  Since  his 
day  the  Liebermann  reagent  (6  per  cent,  potassium  nitrite  in 
cone,  sulphuric  acid)  has  been  extensively  used. 

In  a  subsequent  paper  Liebermann  (35)  describes  how  he 
repeated  De  Luynes's  work  and  found  that  the  action  of  am- 
monia upon  orcinol  gave  him  a  product  part  of  which  was  read- 
ily soluble  in  ammonia.  The  more  insoluble  part  was  dissolved 
in  sodium  hydroxide.  Both  parts  were  now  treated  alike ;  namely, 
acid  was  added  to  each  to  precipitate  the  substance,  which  was 
then  washed,  dissolved  in  alcohol,  and  the  latter  then  evaporated. 
Both  substances  were  found  to  be  amorphous,  and  outwardly 
could  not  be  differentiated.  The  purple  color  obtained  with  alkalis 
showed  a  decided  reddish  tint  with  the  first,  and  a  more  bluish 
one  with  the  second. 

Wurster's  paper  on  "The  formation  of  color  by  means  of 
hydrogen  peroxide"  (36)  is  particularly  worthy  of  close  study. 
He  found  that  in  the  presence  of  hydrogen  peroxide,  ammonia 
and  phenol  gave  a  blue  coloration,  which  gradually  changed  to 
green  and  then  to  yellow.  The  solution  became  quite  decolorized 
when  an  excessive  amount  of  hydrogen  peroxide  was  added. 
The  addition  of  acetone,  alcohol,  or  oxalic  acid  considerably 
hastened  the  reaction.  Hydroxylamine  was  found  to  be  still 
more  effective.  This  substance,  together  with  phenol  and  hydro- 
gen peroxide,  formed  nitroso-phenol,  but  neither  a  blue  nor  a 
green  color  was  evident.  With  the  addition  of  ammonia  the  color 
rapidly  formed.  As  but  a  small  quantity  of  hydroxylamine  was 
necessary,  Wurster  would  explain  this  reaction  by  saying  that 
the  hydroxylamine  is  oxidized  to  nitric  oxide,  which  in  the  solu- 
tion acts  the  part  of  an  oxygen  carrier. 

To  isolate  the  product  Wurster  gives  these  directions :  To 
an  emulsion  of  phenol  in  water  ammonia  is  added  in  such  amount 
a?  to  leave  some  of  the  phenol  still  undissolved.  Some  sodium 
hydroxide  and  an  equal  volume  of  hydrogen  peroxide  are  now 
added,  the  whole  diluted  with  ten  times  its  volume  of  water,  and 
well  shaken.  The  addition  of  a  small  crystal  of  a  salt  of  hydrox- 


27 

ylamine  causes  a  light  blue  color  to  appear  within  a  few  minutes. 
This  soon  changes  to  deep  blue,  and  in  one  or  two  days  becomes 
quite  green.  Without  hydroxylamine  the  color  develops  quite 
slowly,  and  the  maximum  intensity  is  not  reached  before  twenty- 
four  hours,  whereas  with  it  a  full  development  of  color  is  notice- 
able within  a  quarter  of  an  hour. 

By  extracting  the  blue  color  with  ether  part  goes  into  solu- 
tion, giving  a  red  coloration.  A  solution  of  amyl  alcohol  and 
ether  extracts  more  completely. 

As  the  ether  extract  of  the  acid  solution  of  the  dye*  is  red, 
and  the  Liebermann  dye  (nitrous  acid  on  phenol)  under  similar 
conditions  is  yellow,  Wurster  is  inclined  to  believe  that  the  two 
are  quite  different;  and  this  view  receives  support  in  that  their 
spectroscopic  behavior  is  not  the  same. 

Dealing  next  with  the  constitution  of  the  blue  product 
Wurster  states  that  the  whole  behavior  of  the  compound  points 
to  its  identity  with  phenolquinoneimid,  a  substance  first  prepared 
by  Hirsch  (37)  by  the  action  of  quinonechlorimid  on  phenol, 
though  he  could  not  isolate  it. 

Wurster  prepared  phenolquinoneimid  by  adding  ammonia  to 
a  watery  solution  of  quinone  in  the  presence  of  an  excess  of 
phenol.  The  yellow  quinone  solution  quickly  becomes  green  on 
the  addition  of  ammonia,  and  blue  by  stirring  in  contact  with  air. 
A  still  easier  method  is  to  start  with  /-amido  phenol.  By  dis- 
solving this  in  sodium  hydroxide  a  red  solution  is  obtained  which 
becomes  yellow  on  contact  with  air.  By  the  addition  of  phenol, 
phenolquinoneimid  is  at  once  obtained. 

By  studying  other  phenols  Wurster  found  that  all  phenols 
that  have  the  para  position  with  respect  to  the  hydroxyl  group 
unsubstituted  form  quinoneimids.  Where  the  para  position  was 
substituted  the  substances  oxidized  to  a  yellow  product  or  were 
not  attacked  at  all. 

The  author  concludes  significantly  by  saying  that  since  it 
has  been  shown  that  both  ammonia  and  phenol  are  decomposi- 
tion products  of  proteins,  the  formation  of  color  in  plants  bears 
a  certain  relationship  to  the  hydrogen  peroxide  that  is  present. 

Zulkowski  and  Peters  (38)  carefully  repeated  Liebermann's 
*  Here  the  word  is  rather  loosely  employed. 


28 

work  on  the  action  of  ammonia  upon  orcinol.  By  allowing  a 
mixture  of  50  gms.  orcin  in  200  c.c.  water  and  200  c.c.  ammonium 
hyclroxid  solution  [strength  not  given]  to  stand  for  two  months  a 
thick  jelly  was  obtained,  from  which  the  following  three  colored 
substances  were  isolated : 

1)  A  red-colored  product,  orcein,  obtained  in  microscopi- 
cal crystals  from  a  mixture  of  water  and  alcohol.     With  alcohol 
it  gives  a  carmine-red  solution,  and  with  ammonia,  the  fixed 
alkalis,  and  the  alkaline  carbonates,  a  bluish-violet.     Insoluble 
in  water.     Yield,  50%. 

2)  A  yellow  crystalline  substance,  soluble  in  ether  and  al- 
cohol, and  less  so  in  boiling  water,  in  each  case  giving  a  yellow 
solution. 

3)  An  amorphous,  litmus-like  substance,  with  a  greenish 
metallic-luster.     Insoluble  in  alcohol,  soluble  in  alkalis  to  a  dark 
blue  solution,  which  turns  red  on  the  addition  of  acids. 

By  the  addition  of  hydrogen  peroxide,  the  rapidity  of  the 
process  could  be  greatly  increased.  What  required  days  before 
could  be  accomplished  in  so  many  hours. 

Under  otherwise  identical  conditions  resorcinol  did  not 
yield  an  orcei'n-like  substance ;  but  the  latter  could  be  obtained 
by  allowing  orcinol  (142  parts),  resorcinol  (110  parts),  22,% 
ammonium  hydroxide  solution  (7.7  p.),  and  3%  iI2O2  (3  MM)  p.) 
to  stand  for  several  days.  By  recrystallizing  the  product  from 
alcohol  a  bronze  lustrous  substance  was  obtained,  the  red  acetic 
acid  solution  becoming  blue  upon  the  addition  of  ammonia  or 
alkali. 

Maseau  (39)  in  a  study  of  the  comparative  color  reactions 
of  the  phenols  with  ammonia  and  iodine  in  the  presence  of  alco- 
hol, describes  the  colors  with  ammonia  as  follows :  Pyrocate- 
chin,  reddish-brown ;  hydroquinone,  yellow ;  pyrogallol,  black- 
ish-brown ;  orcin,  red  to  violet.  Phenol,  resorcinol  and  naphthol 
remained  colorless. 

It  is  quite  apparent  that  the  author  did  not  take  the  time 
element  into  consideration. 

THE  ACTION  OF  AMMONIA  ON  THYMOL. 

The  brilliant  Lallemand,  in  his  exhaustive  study  of  thymol, 
refers  to  the  action  of  ammonia  upon  it  in  no  uncertain  terms. 


29 

"Le  thymol,"  he  \vrites  (10),  "n'est  pas  altere  par  1'ammoniaque 
liquide ;  mais  il  dissout  une  grande  quantite  de  gaz  ammoniac 
qu'il  abandonne  lentement  en  se  solidifant."  This  view  became 
the  accepted  one.  We  find  Watt,  for  example,  quoting  it  in  his 
dictionary  (40).  Just  as  in  many  another  reaction  a  neglect  of 
the  time  element  failed  to  give  any  visible  result. 

The  first  one  to  record  any  reaction  is  Lex  (30).  In  de- 
scribing some  new  color  tests  for  phenol,  the  author  records 
that  the  addition  of  ammonia  to  phenol  causes  a  blue  color  to 
form  under  either  one  of  the  following  conditions :  a)  Warm- 
ing the  solution  with  bleaching  powder;  b)  warming  with  ba- 
rium peroxide;  or  c)  allowing  to  stand  exposed  to  the  air. 
He  adds,  in  a  casual  note,  that  he  finds  thymol  to  behave  simi- 
larly.* 

In  a  comparative  study  of  the  behavior  of  thymol  and 
phenol  with  various  reagents,  Hirschsohn  (41)  found  that  when 
thymol  (1:1000)  was  heated  with  bleaching  powder  and  am- 
monia the  solution  became  cloudy,  and  after  a  time  showed  a 
greenish  tinge.  In  a  concentration  of  1  :  2000  and  I  :  4000  only 
a  cloudiness  was  obtained.  The  substitution  of  chlorine  water 
for  bleaching  powder  produced  a  bluish-green  coloration. 

Wurster,  in  the  article  already  quoted  at  some  length  (36) 
showed  that  thymol,  in  the  presence  of  hydrogen  peroxide  and 
ammonia,  forms  a  quinoneimid  analogous  to  that  obtained  from 
phenol.  The  acid  properties  of  the  resulting  imid — a  red  oil 
insoluble  in  water — were  found  to  be  so  slight  that  dilute  am- 
monia had  no  tendency  to  salt  formation.  The  blue  sodium 
salt  could  be  decomposed  with  a  large  quantity  of  water,  but 
the  potassium  salt  was  more  stable. 

*  The  casual  reference  to  thymol  comes  in  the  last  three  lines  of  the 
article.  The  quotation  in  full  is  as  follows:  "Uebrigens  zeigt  das  einzige 
fernere  Glied  der  Ph-enolreihe,  welches  ich  zu  priifen  Gelegenheit  hatte. 
das  thymol,  insofern  ein  ganz  analoges  Verhalten."  That  this  observa- 
tion attracted  no  attention  is  seen  by  the  fact  that  no  mention  of  it  wliat- 
ever  w  to  be  found  in  She  literature.  It  was  only  by  the  merest  chance 
that  we  came  across  this  article,  and  then  long  after  the  experimental  part 
of  this  research  had  been  begun.  However,  even  had  we  stumbled  across  it 
at  the  very  beginning  of  this  inquiry  Lex's  comment  would  merely  have 
given  added  impetus  to  our  desire  to  inaugurate  this  investigation. 


30 

From  the  title  of  his  essay — "The  role  of  hydrogen  perox- 
ide in  the  formation  of  color" — we  not  only  get  the  object  of 
this  research,  but  also  the  author's  opinion  that  the  oxidizing 
agent  plays  an  indispensable  part  in  the  formation  of  the  color. 
This  is  emphasized  more  than  once  in  the  contents. 

In  a  recent  communication,  Gies  (42),  in  following  up  his 
previous  observations  (1),  shows  that  filter  paper  soaked  in 
the  blue,  alkaline,  alcoholic  solution  (obtained  from  the  blue 
of  an  ammonium  hydroxide-thymol  mixture  by  extracting  the 
color  with  ether,  evaporating  the  latter,  dissolving  in  alcohol,  and 
rendering  slightly  alkaline),  and  then  dried  at  room  tempera- 
ture, "assumes  a  bright  red  color  as  the  alcohol  disappears. 
Treated  with  alcohol,  such  red  filter  paper,  particularly  if 
slightly  moist,  becomes  bright  green."  Interesting  probabilities 
suggested  by  these  results,  and  the  possible  relationship  of  these 
color  phenomena  to  the  pigments  in  the  Monardas*  and  other 
plants,  will  be  investigated. 

IS  THE  BLUE  COLOR  DUE  TO  AN   IMPURITY? 

The  first  question  that  suggested  itself  was  whether  the 
blue  color  was  due  to  some  impurity  that  was  present  in  the 
thymol?  If  so,  the  probabilities  were  that  the  same  amounts 
of  different  varieties  of  thymol  would  show  marked  differences 
in  intensity  of  color.  Three  varieties  on  the  market,  Merck's, 
Eimer  and  Amend's,  and  Kahlbaum's,  were  procured,  but  no 
differences  could  be  detected  (Table  I). 

In  order  to  eliminate  further  doubt,  it  was  decided  to  purify 
the  thymol.  After  several  preliminary  experiments,  a  conven- 

*  Wakeman :  Bulletin  of  the  Univ.  of  Wisconsin,  No.  448 ;  Science 
series,  1911,  IV,  p.  25.  Colored  pigments  in  the  corollas  of  Monardas 
didyma,  fistulosa,  and  punctata,  are  described.  These  are  regarded  as 
probable  oxidation  products  of  the  thymol  and  carvacrol.  Hydrothymo- 
quinone,  thymoquinone,  and  dihydroxythymoquinone,  have  been  isolated. 
These  are  known  to  form  colored  compounds  by  combining  with 
monatomic  phenols. 


lent  means  of  doing  this  was  found  to  be  to  steam  distil  the 
substance,  and  recrystallize  the  product  from  glacial  acetic  acid.* 

As  tests  of  purity,  the  melting  points  of  the  different  sam- 
ples were  compared.  However,  even  here  one  is  beset  with  diffi- 
culties, for  in  the  literature  melting  points  ranging  anywhere 
from  44°  to  53°  are  to  be  found.f  With  a  view  to  explaining 
some  of  these  differences,  slight  modifications  in  procedure  were 
adopted  at  different  times  (Table  II).  The  last  determination 
(number  8),  giving  the  melting  point  from  50-50.5°  should  be 
taken  as  the  most  reliable. 

From  a  glance  at  the  table  it  will  be  seen  that  the  melting 
points  of  the  different  samples  agree  remarkably  well.  This 
shows  that  the  samples  obtained  from  Merck,  Eimer  £  Amend, 
and  Kahlbaum,  were  equally  pure,  and  that  steam  distillation 

*  In  some  of  these  steam  distillations  the  thymol  came  over  in  the 
form  of  a  colorless  oil;  by  merely  transferring  into  another  flask  the  oil 
would  solidify  into  a  shining  white  mass.  This  solidification  by  mere 
agitation  is  a  marked  characteristic,  and  has  been  noticed  by  many 
workers. 

If  the  condenser  is  too  cold  the  thymol  very  readily  solidifies  in  the 
tube.  A  little  alcohol  or  acetic  acid  will  easily  dissolve  this. 

As  this  phenol  is  exceedingly  soluble  in  glacial  acetic  acid,  and  as 
crystallization  will  not  take  place  from  a  too  dilute  solution,  quantities 
of  the  steam  distilled  thymol  were  added  to  a  small  quantity  of  acetic 
till  an  almost  saturated  solution  was  obtained  (by  the  aid  of  gentle  heat). 
Then  one  or  two  c.c.  more  of  the  acid  were  added. 

Thymol  crystallizes  from  glacial  acetic  in  large,  colorless,  hexa- 
gonal plates,  a  mosaic  of  the  crystals  appearing  on  the  surface. 

In  the  course  of  these  crystallizations  an  interesting  means  of  study- 
ing the  development  and  formation  of  crystals  was  hit  upon.  This  method 
consisted  in  very  slowly  pouring  an  almost  saturated  solution  of  thymol 
in  glacial  acetic  acid  into  a  large  quantity  of  water.  Soon  small 
droplets  appear,  and  in  these  latter  the  nucleus  of  the  crystal,  in  the  s'hape 
of  a  speck  of  white  solid,  springs  into  being.  This  enlarges  and  spreads 
until  the  crystal  is  formed. 

f  Carnelly,  in  his  "Melting  and  Boiling  Point  Tables"  (1885),  I,  224, 
gives  the  following:  Liquid  (Febve)  ;  44°  (Lallemand,  Sten'house, 
Widman)  ;  46°  (Kekule  and  Fleischer);  48°  (Arppe)  ;  49.2°  (Schiff)  ; 
51°  (Andresen)  ;  53°  (Haines). 

Fehling  ("Handworterbuch  der  Chemie,"  7,  969  (1905)  ),  supple- 
ments this  by  giving  Mentschutkin's  (50°)  and  Reinsert' s  (49.7°)  figures. 


32 

and  recrystallization  did  not  tend  to  increase  the  purity.  Above 
all,  the  purified  products  reacted  with  ammonia  in  precisely  the 
same  way,  and  to  precisely  the  same  extent  as  the  non-purified 
materials. 

TABLE  I. 

Comparison  of  intensity  of  color  for  different  samp-es  of 

thymol. 

ABC 

Thymol    0.5  gm.  0.5  gm.  0.5  gm. 

Ammonia    (10%)*    100  c.c.  100  c.c.  IGOc.c. 

Alcohol  (95%)* 10  c.c.  10  c.c.  10  c.c. 

A— Merck  product. 

B — Eimer  &  Amend  product. 

C — Kahlbaum  product. 
Conclusion. — Intensity  about  same  in  all. 

TABLE  II. 

Determination   of   the   melting   point   of    the   ordinary   and 
purified  thymol.     [The  numbers  refer  to  °C.] 

Merck's     Merck's  steam 

„.          p     .          ,     v  ,  ,,  steam  distil,  and 

Merck  Eimer  &  Amend     Kahlbaum     d;stjllej          recryst. 

from  acetic 

1.  48  47-48  47-48 

2.  48  47-48  48 

3.  49+  48  49 

4.  49+  (0.2-0.3)  49  49 

5.  49 +(0.4-0.5)     49 

6.  48  49 

7.  48.5  48.5  49 

8.  50-50.5  50-50.5  50.5  50.5  50.5 
Notes. — Roth's  Melting  Point  Apparatus  was  used  [see  Ber. 

d.  D.  Chem.  Gesell.  19,  19TO  (1886)].  Melting  points  were 
taken  only  after  complete  fusion. 

Note  on  the  method  of  drying  thymol:  It  was  noticed  that  when 
concentrated  sulphuric  acid  was  used  as  a  drying  agent  in  the  desiccator 
the  acid  gradually  changed  to  a  brownish-red  color,  and  fine  violet  colored 
deposits  were  repeatedly  obtained.  This  result  was  always  obtained  in 
the  presence  of  thymol.  Calcium  chloride  was  therefore  substituted. 

*  Unless  otherwise  stated  the  ammonia  and  alcohol  used  throughout 
are  respectively  10%  and  95%.  Alcohol  was  used  on  the  supposition  that 
it  favored  the  reaction  by  increasing  the  solubility  of  thymol.  See  page  44. 


33 

CERTAIN    QUANTITATIVE    RELATIONSHIPS    BETWEEN     THYMOL,    AM- 
MONIA  AND  ALCOHOL,   AND  THYMOL   AND   AMMONIA   ALONE. 

Having  shown  that  the  blue  color  is  not  due  to  an  impurity 
in  the  thymol,  it  became  important  now  to  establish  definite 
quantitative  relationships  in  order  to  determine  to  what  extent 
the  different  reagents  took  part  in  the  reaction.  Since  we  were 
here  dealing  with  three  factors,  thymol,  ammonia  and  alcohol, 
the  most  logical  method  that  suggested  itself  was  to  conduct 
experiments  on  the  basis  of  one  variable  and  two  constants. 
Table  III  shows  that  the  intensity  of  the  color  is  proportional  to 
the  amount  of  thymol  present,  but  Tables  IV  and  X  show 
that  beyond  certain  limits  this  does  not  hold.  In  the  same  way 
it  was  shown  that  the  intensity  of  the  color  varied  directly  with 
the  amount  of  ammonia  present  (Table  V),  and  subsequently, 
that  more  concentrated  solutions  of  ammonia  inhibited  the  for- 
mation of  color. 

The  action  of  alcohol  was  most  peculiar.  It  was  at  first 
supposed,  from  the  original  experiments  on  elastin  (1),  that 
alcohol  would  considerably  accelerate  the  reaction.  But  this  did 
not  prove  to  be  the  case  (Table  VI)  ;  and  indeed,  under  certain 
conditions,  it  acted  as  a  retarding  agent  (Table  VII). 

Of  course,  this  at  once  suggested  the  idea  of  dispensing 
with  the  alcohol  altogether,  and  most  satisfactory  results  were 
obtained  (Table  VIII).* 

*  One  of  the  most  interesting  phenomena  in  these  observations  was 
the  behavior  of  thymol  when  added  to  the  ammoniacal  solution.  As  soon 
as  the  powdered  thymol  touched  the  surface  of  the  liquid  it  tended  to  form 
globules.  This  was  particularly  marked  when  the  quantities  of  thymol 
added  were  comparatively  large.  When  a  globule  had  reached  a  certain 
size  it  would  sink  to  the  bottom.  Upon  standing  the  globule  would  grad- 
ually assume  a  reddish  or  violet  tint,  and  a  pear-shaped  form.  Two  op- 
posing forces  now  came  into  play, — the  upper  liquid  portion  of  the  globule 
which  tended  to  force  its  way  upward,  and  the  lower,  which  restrained  it. 
The  upper  portion  gradually  increased  in  size,  and  after  it  had  attained 
a  certain  volume  it  broke  away  from  the  rest  of  the  globule  and  came  to 
the  surface,  forming  colored  (usually  violet)  oily  layers.  A  repetition 
of  the  above  phenomenon  would  now  commence  in  the  globule  at  the 
bottom  ;  and,  indeed,  this  would  continue  for  several  days,  till  finally  the 
entire  globule  had  disintegrated.  This  might  offer  an  interesting  field  for 
physico-chemical  study. 


34 

Under  certain  conditions  water  was  found  to  take  an  inter- 
esting part  in  the  formation  of  color  (Table  XI). 

With  regard  to  the  delicacy  of  the  reaction,  it  was  found 
that  within  five  days  a  distinct  coloration  was  obtained  in  a 
concentration  of  1:25,000  (Table  IX). 

TABLE  III. 

Effect  of  varying  thymol,  with  ammonia  and  alcohol  constant. 
12345 

Thymol   0.1  gm.      0.2  gm.      0.3  gm.      0.4  gm.      0.5  gm. 

Ammonia    (10%)    100  c.c.       100  c.c.       100  c.c.       100  c.c.       100  c.c. 

Alcohol   (95%)    10  c.c.        10  c.c.        10  c.c.        10  c.c.        10  c.c. 

Conclusion. — Intensity  of  color  varies  directly  with  the 
amount  of  thymol  present. 

Notes. — Color  noticeable  one  hour  after  commencing.  Color 
gradually  changes  from  green  to  blue,  the  change  taking  four, 
and  perhaps  more  days. 

TABLE  IV. 

What  amount  of  thymol  gives  the  maximum  color? 

12345678 
Thymol    ..0.02gm.  0.05  gm.  0.5  gm.  0.6  gm.  0.7  gm.  0.8  gm.  0.9  gm.  1.00  gm. 
Ammonia.    100  c.c.     100  c.c.  100  c.c.  100 c.c.  100  c.c.  100 c.c.  100  c.c.     100 c.c. 
Alcohol...      10  c.c.      10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c.      10  c.c. 

Conclusion. — Three  gives  maximum  color. 

Notes. — One  showed  first  trace  of  color  in  \l/2  hours.  After 
46  hours  the  color  was  still  a  very  light  green. 

Intensity  of  colors  in  4  and  5  about  same  as  in  3,  but 
pinkish  globules  on  top. 

In  6  color  somewhat  less  intense  than  5 ;  7  and  8  showed 
faintest  trace  of  color  after  1^  hours,  but  solution  was  very 
cloudy.  Pink  globules  on  top.  After  46  hours'  standing  the  in- 
tensity of  the  blue  color  showed  but  very  slight  sign  of  increase. 

TABLE  V. 

Effect  of  varying  ammonia,  with  thymol  and  alcohol  constant. 
12  34  5678  9 

Thymol — 

0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm. 


35 

TABLE  V — Continued. 

Ammonia — 

lOc.c.    20c.c.    30c.c.    40c.c.    50  c.c.    60  c.c.    70  c.c.    SOc.c.    90c.c. 
Water— 

90  c.c.    80  c.c.    70  c.c.    60  c.c.     SOc.c.    40  c.c.    30  c.c.    20  c.c.     10  c.c. 
Alcohol — 

10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c.     10  c.c    10  c.c. 

Conclusion. — Intensity    of    color    varies    directly    with    the 
amount  of  ammonia  present. 

Notes. — After  26  hours,  large  crystals — partly  pinkish,  partly 
colorless — separated  out  in  1. 

After  41  hours'  standing  the  solution  in  2  changed  to  pink. 

After  91  hours'  1  and  3  were  slightly  pink. 

1  and  2  were  repeated,  and  beyond  getting  smaller  crystals 
in   1,  identical  results  were  obtained. 

TABLE  VI. 

Effect  of  varying  alcohol,  with  thymol  and  ammonia  constant. 
123456789  10 

Thymol— 

0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gtn.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm . 
Ammonia — 

100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100 c.c.  100  c.c.  100  c.c. 
Alcohol — 

1  c.c.     2  c.c.     3  c.c.     4  c.c.     5  c.c.     6  c.c.     7  c.c.    8  c.c.     9  c.c.    10  c.c. 
Water— 

10  c.c.     9  c.c.     8  c.c.     7  c.c.     6  c.c.     5  c.c.     4  c.c.     3  c.c.      2  c.c.      1  c.c. 
Conclusion. — Intensity  of  color  about  the  same  in  all. 
Notes. — No   difference  in   intensity  could   be  noticed  even 
after  22  hours. 

TABLE  VII. 

Effect  of  variation  of  alcohol  upon  a  comparatively  large 
quantity  of  thymol  in  the  presence  of  a  constant  quantity  of 

ammonia. 

123456 

Thymol    1  gm.      1  gm.      1  gm.      1  gm.      1  gm.      1  gm.      1  gm. 

Ammonia  (10%)    100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c. 
Alcohol    (95%).     10  c.c.    20  c.c.    30  c.c.    40  c.c.    SOc.c.    60  c.c.    70  c.c. 

Water    70  c.c.    60  c.c.    SOc.c.    40  c.c.    30  c.c.    20  c.c.     10  c.c. 

Conclusion. — Intensity  of  color  decreases  with  increase  of 
alcohol. 


36 

Notes. — The  difference  in  results  between  this  experiment 
and  the  one  above  is  probably  due  to  two  causes:  1)  Greater 
quantity  of  thymol  used;  2)  greater  differences  in  amount  of 
alcohol. 

Purplish  drops  in  1,  and  to  a  less  degree  in  2  and  3;  4 
hardly  any;  5,  6,  7  none  at  all.  This  is  due  to  the  fact  that  the 
more  alcohol  present,  the  more  perfect  the  solution. 

It  has  been  noticed  that  whenever  there  is  more  thymol  than 
will  dissolve,  the  excess  tends  to  change  to  a  pinkish  color — 
sometimes  appearing  in  the  form  of  a  pinkish  precipitate,  more 
often  as  pinkish  or  purplish  globules. 

TABLE  VIII. 

Effect  of  varying  ammonia  in  the  presence  of  a  constant 
quantity  of  thymol  and  in  the  absence  of  alcohol. 

12  345  678  9 

Thymol — 

0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm.  0.5  gm. 
Ammonia  (10%) — 

10  c.c.    20c.c.    30  c.c.    40c.c.    50  c.c.    60  c.c.    70c.c.    SOc.c.    90c.c. 
Water- 

90  c.c.    SOc.c.    70  c.c.    60  c.c.     SOc.c.    40  c.c.    30  c.c.    20  c.c.     10c.c. 

Conclusion. — Intensity  of  color  varies  directly  with  the 
amount  of  ammonia  present. 

Notes. — The  color  is  obtained  as  well  without  alcohol  as 
with  it. 

1  began  to  show  a  faint  trace  of  color  only  after  4  hrs. 
(In  the  presence  of  10  c.c.  alcohol  the  same  sample  gave  evi- 
dence of  color  in  ll/2  hrs.) 

1,  2,  3,  4,  5  contained  crystalline  precipitates  (probably  un- 
changed thymol),  the  quantity  decreasing  from  1-5.  6,  7,  8,  9  did 
not  show  this. 

After  39  hours,  solution  in  1  became  pink ;  2  also,  but  to  a 
less  extent. 

(1  was  repeated  and  the  pink  color  verified.) 


37 

TABLE  IX. 

How  small  a  quantity  of  thymol  is  necessary  to  produce 
the  blue  color? 

1234 

Thymol  (saturated       10  c.c.  15  c.c.  20  c.c.  25  c.c. 

solution  in  water)  (=0.008gm.)    (=0.012gm.)    (=0.016gm.)   (=0.02gm.) 
Ammonia    (20%).     100  c.c.  100  c.c.  100  c.c.  100  c.c. 

Water   , 90  c.c.  85  c.c.  80  c,c.  75  c.c. 

[2  :  50,000]        [3  :  50,000]     [4  :  50,000]     [5  :  50,000] 

Conclusion. — After  five  days  1  became  faint  green. 

Notes. — 20%  ammonia  was  used  as  the  added  volumes  of 
water  and  thymol  solution  reduced  the  solutions  to  10%. 

TABLE  X. 

Effect  of  ammonia  upon  comparatively  large  quantities  of 
thymol. 

1  2  3 

Thymol    1  gm.  2  gm.  5  gm. 

Ammonia    100  c.c.  100  c.c.  100  c.c. 

Conclusion. — Excess  of  thymol  tends  to  change  the  blue  color 
of  the  solution  to  red,  purplish  layers  appearing  at  the  surface. 

Notes. — Turbidity  increases  with  increase  of  thymol.  Red- 
dish drops  on  top  increase  with  increase  of  thymol. 

After  1  hour  1  began  to  show  the  usual  greenish  tinge, 
but  neither  2  nor  3  showed  any  color,  though  they  were  both 
very  turbid. 

In  20  hrs.  1  had  become  blue,  2  bluish-red  and  3  almost 
wholly  red.  In  all  reddish  oily  drops  appeared  at  the  surface — 
more  so  in  3,  less  in  1. 

In  5  days  all  the  samples  had  become  more  blue.  (It  has 
been  noticed  that  whenever  the  red  is  more  pronounced  at  first 
it  has  a  tendency  gradually  to  change  to  the  blue.) 

After  2  weeks,  the  blue  was  more  marked  than  ever.  The 
solutions  were  all  bluish,  the  oily  drops  on  top  purplish. 

TABLE  XI. 

Effect  of  different  quantities  of  water  upon  a  relatively  large 

amount  of  thymol. 

123456 

Thymol    1  gm.     1  gm.     1  gm.     1  gm.     1  gm.     1  gm.     1  gm. 


38 

TABLE  XL — Continued. 

Ammonia  (10%)...  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100  c.c.  100 c.c. 
Alcohol  (95%)  ....  10  c.c.  10  c.c.  10  c.c.  10  c.c.  10  c.c.  10c.c.  10c.c. 
Water  Oc.c.  10  c.c.  20  c.c.  30  c.c.  40  c.c.  50  c.c.  60  c.c. 

Conclusion. — 1  is  very  turbid  and  has  pinkish  tinge;  2,  3, 
4,  5,  6,  7,  all  blue.  The  10  c.c.  difference  in  water  in  1  and  2 
plays  an  important  part. 

Notes. — Above  was  undertaken  because  thym.  1 ;  am.  100  ;  ale. 
10;  water  0,  showed  hardly  a  trace  of  blue  color,  but  decided 
turbidity,  and  pinkish  tinge  in  solution ;  whereas  thym.  1 ;  am. 
100 ;  ale.  10 ;  water  70,  showed  the  usual  greenish-blue. 

ACCELERATIN^,     INDIFFERENT     AND     RETARDING     AGENTS     IN      THE 
FORMATION   OF  THE  BLUE   COLOR. 

Quantitative  relationships  having  been  established,  it  now  be- 
came advisable  to  investigate  other  factors  that  might  influence 
color  formation. 

It  was  found  that  heating  on  the  water  bath  5-10  minutes 
accomplished  what  standing  at  room  temperature  would  have 
taken  24  hrs.  or  more.*  (Table  XII.)  On  the  other  hand,  sur- 
rounding a  mixture  of  ammonia  and  thymol  with  ice  prevented 
the  formation  of  the  color  altogether.  (Table  XIII.) 

Bearing  in  mind  the  effect  light  has  upon  phenol  (43)  as 
well  as  on  quinone  and  thymoquinone  (44)  t,  it  became  desirable 
to  ascertain  what  would  happen  in  the  absence  of  sunlight ;  but 
this  did  not  effect  the  color  formation  in  the  least  (Table  XIV). 

The  effect  of  the  addition  of  a  small  quantity  of  ether  was 
rather  unexpected :  not  a  trace  of  color  could  be  detected  after 
20  hours'  standing  (Table  XV). § 

*  It  may  be  remarked  here  that  in  most  of  the  subsequent  work  heat 
was  not  resorted  to.  Continued  heating  would  tend  to  change  the  blue 
color  into  red,  but  the  blue  could  be  restored  by  adding  more  ammonia. 

f  At  this  time  the  theory  had  been  formed  that  thymoquinone  is  an 
intermediate  product  in  the  formation  of  the  blue  color. 

§  To  explain  this  one  or  two  points  may  be  suggested.  In  the  first 
place,  ether  and  ammonia  are  not  very  miscible,  and  thymol  is  exceeding- 
ly soluble  in  ether.  Then,  again,  the  ether,  which  consists  of  the  upper 
portion  of  liquid,  prevents  to  a  certain  extent  the  ingress  of  air;  and  air, 
as  will  be  shown  later,  is  an  essential  factor. 


39 

TABLE  XII. 
Effect  of  heat  upon  the  production  of  the  blue  color. 

Thymol    0.5  gm. 

Ammonia    ( 10% )     100  c.c. 

Alcohol    (95%)    10  c.c. 

Conclusion. — Color  well  marked  within  5  minutes.  With- 
in 10  minutes  the  color  is  so  intense,  that  a  sample  after  stand- 
ing 24  hrs.  cannot  compare  with  it. 

Note. — Heating  was  accomplished  on  a  boiling  water  bath, 
under  reflux. 

TABLE  XIII. 

Effect  of  cold  upon  the  production  of  the  blue  color. 

Into  a  glass-stoppered  cylinder  were  placed  0.5  gm.  thymol 
and  150  c.c.  ammonium  hydroxid  solution.  This  was  surrounded 
by  ice. 

Conclusion. — Cold  prevents  formation  of  blue  color. 

Notes. — No  trace  of  color  at  the  end  of  5^  hrs.,  though 
a  control  (containing  the  same  quantities,  but  exposed  to  room 
temperature),  had  become  colored  in  the  usual  manner.  The 
crystals  of  thymol  were  found  quite  unaffected. 

TABLE  XIV. 

Effect  of  ammonia  upon  thymol  in  absence  of  sunlight. 
0.5  gm.  Thymol     1      Put    into    dark    bottle,    then 
100  c.c.  Ammonia  rinto   desk   locker,  and  allowed 
10  c.c.  Alcohol     J  to  stand. 

Conclusion. — Absence  of  sunlight  does  not  prevent  the  for- 
mation of  color. 

Notes. — After  2  hours  a  part  of  the  solution  was  poured  into 
a  transparent  bottle,  and  it  was  found  to  have  turned  green.  It 
continued  to  change  more  towards  the  blue,  in  every  way  con- 
forming to  that  found  in  other  experiments. 

TABLE  XV. 

Effect  of  ether  upon  thymol  in  the  presence  of  ammonia. 

Thymol    (about)   0.1  gm. 

'  Ammonia    50  c.c. 

Ether    5  c.c. 

Conclusion. — Ether  prevents  the  formation  of  the  color. 


4o 

Notes. — After  20  hours  no  change  could  be  noticed.  Alcohol 
was  then  added.  3  days  later  a  slight  pink  was  noticed  in  the 
emulsion  which  had  formed  on  top.  For  3  successive  days 
more  alcohol  was  added  each  day,  but  no  further  change  was 
visible. 

At  the  end  of  11  days  the  solution  showed  a  decided  green- 
ish tinge,  and  the  pink  color  in  the  emulsion  was  very  clear. 

ARE  AMMONIA   AND   THYMOL  ALONE  THE   ACTIVE   FACTORS   IN   THE 
FORMATION  OF  THE  BLUE  COLOR? 

Liebermann,  in  his  important  paper  upon  the  action  of  ni- 
trous acids  on  phenols  [to  which  reference  has  already  been 
made  (34)  ],  tells  us  what  led  him  to  this  color  test. 

It  had  been  found  that  ammonia  in  the  presence  of  air 
converts  orcin  into  orcein,  and  it  did  not  seem  improbable  to 
him  that  the  ammonia  and  the  oxygen  of  the  air  combined  to 
form  nitrous  acid  and  water.  If  that  were  so,  then  nitrous  acid 
could  be  substituted.  When  nitrous  acid  was  tried,  a  colored 
substance  was  readily  obtained,  though  this  did  not  prove  to  be 
orcein.* 

Liebermann's  illuminating  work  suggested  that  possibly  in 
our  own  case  the  oxygen  of  the  air  plays  a  role  in  the  reaction. 
The  formation  of  the  blue  color  should  therefore  be  prevented 
by  preventing  ingress  of  air,  or  replacing  air  by  another  gas ; 
and  should  be  hastened  by  passing  a  rapid  stream  of  air  through 
the  solution,  or  adding  a  suitable  oxidizing  agent. 

Several  preliminary  qualitative  experiments  were  made.  The 
simple  method  of  placing  a  beaker  containing  ammonia  and 
thymol  into  a  desiccator  and  exhausting  the  air,  has  the  objec- 
tion that  by  so  doing  most  of  the  ammonia  is  also  driven  off. 
The  method,  again,  of  filling  to  overflowing  a  bottle  with  am- 
monia and  thymol  and  then  stoppering  it,  also  has  the  objection 
that  all  air,  either  dissolved  or  otherwise,  cannot  be  excluded. 
To  overcome  this,  thymol  and  ammonia  were  treated  with  hy- 
drogen gas  for  ten  minutes,  placed  in  a  tight-stoppered  bottle, 
put  in  a  desiccator,  and  the  air  exhausted.  After  23  hours' 
standing,  a  pale  green  color  formed;  but  the  lack  of  intensity 
*  "Dieser  Farbstoff  ist  jedoch  kern  Orcein,  sondern  ein  neue  Substanz." 


of  color  clearly  suggested  that  the  exclusion  of  air  does  have 
an  inhibitory  influence,  and  that  the  ultimate  appearance  of 
color  was  due  to  air  which  had  found  its  way  in.  At  the  same 
time,  this  experiment  demonstrated  how  very  readily  even  a  very 
small  quantity  of  air  would  suffice  to  produce  the  color. 

Another  method  was  to  pass  hydrogen  for  a  few  minutes 
through  a  20%  solution  of  ammonia  (20%  because  much  of  the 
ammonia  is  lost  by  evaporation),  pour  this  directly  into  a  bot- 
tle containing  thymol  through  which  hydrogen  had  also  been 
passed,  and  continue  passing  the  gas  through  the  mixture  for 
several  more  minutes.  The  bottle  was  now  stoppered  and  al- 
lowed to  stand  beneath  an  inverted  funnel  connected  with  a 
hydrogen  generator.  After  three  hours  no  trace  of  color  could 
be  noticed. 

A  still  more  efficient  means  of  preventing  the  formation  of 
color,  and  one  which  later  led  to  fruitful  results,  was  to  evolve 
hydrogen  within  the  thymol-ammonia  mixture  by  adding  zinc 
dust. 

From  theoretical  conceptions,  and  as  a  result  of  the  inhibi- 
tory influence  of  the  absence  of  air  upon  the  formation  of  color, 
it  was  argued  that  an  induced  current  of  air  should  accelerate 
color  production.  Here  the  valid  objection  can  be  raised  that 
by  passing  air  through  the  solution,  much  of  the  ammonia  is  lost, 
and  as  the  intensity  of  the  color  varies  with  the  amount  of  am- 
monia present,  it  would  be  almost  impossible  to  arrive  at  any 
definite  conclusion.  However,  by  causing  the  current  of  air  to  pass 
through  several  ammonia  solutions  first,  and  then  through  the 
solution  tested,  the  difficulty  was  overcome;  and  it  soon  became 
apparent  that  under  these  circumstances  the  color  forms  sooner 
than  by  merely  allowing  the  ammonia  thymol  mixture  to  stand. 

Comparative  effects  of  passing  air  and  hydrogen,  and  add- 
ing hydrogen  peroxide,  to  thymol-ammonia  mixtures,  were  made 
(Tables  XVI  and  XVII).  From  these  and  from  foregoing  ex- 
periments we  were  justified  in  concluding  that  the  oxygen  of  the 
air  is  a  vital  factor  in  the  ammonia-thymol  reaction.  The  fact 
that  hydrogen  peroxide  very  considerably  increased  the  rapidity 
of  formation  of  color  was  further  evidence  of  the  important 
part  oxygen  played. 


42 
TABLE  XVI. 

Comparison  of  effects  induced  by  passing  currents  of  air  and 
hydrogen  through  ammonia-thymol  mixture. 

1   (air)          2  (hydrogen)       3  (control) 

Thymol  0.5  gm.  0.5  gm.  0.5  gm. 

Ammonia  (20%)    100  c.c.  100  c.c. 

Ammonia  (10%)    100  c.c. 

(Used  20%  ammonia  because  much  of  it  was  removed.) 

Conclusion. — Replacing  air  by  hydrogen  prevents  formation 
of  color. 

Notes. — Through  1  a  current  of  air  was  passed,  through  2 
a  current  of  hydrogen,  the  rate  of  flow  having  been  so  regulated 
as  to  be  as  nearly  as  possible  the  same  (16  bubbles  in  10  sec- 
onds). 

Commenced  at  11 : 15  a.  m. 

2  p.  m. — Put  all  3  solutions  into  bottles  of  equal  size,  and 
compared  colors. 

1.  Greenish-blue.  2.  No  color.  3.  Greenish-blue.  The 
mere  transference  from  one  bottle  to  another  was  apparently 
enough  to  give  2  the  necessary  amount  of  oxygen;  for  when 
the  process  was  now  continued,  and  3^  hours  later  color  compari- 
sons were  once  again  made,  2  showed  a  VERY  faint  green,  the 
other  two  being  quite  bluish. 

TABLE  XVII. 

Comparison  of  action  of  air  (current),  hydrogen  and  hydro- 
gen peroxide  on  ammonia-thymol. 

1  234 

(control)    (hydrogen  (air  cur-  (hydrogen) 

peroxide)        rent) 

Thymol  0.5  gm.  0.5  0.5  0.5 

Ammonia    (20%)    100  c.c.       100  c.c. 

Ammonia    (10%)     100  c.c.  100  c.c. 

Hydrogen  peroxide  (3%)  10  c.c. 

Conclusions. — Replacement  of  air  by  hydrogen  prevents  for- 
mation of  color. 

Hydrogen  peroxide  increases  rapidity  of  formation  of  color 
— another  proof  of  how  important  a  part  oxygen  plays  in  this 
reaction. 


43 

Notes.— A  distinct  green  was  produced  in  2  in  10-15  min- 
utes, a  bluish-green  in  25  minutes,  whereas  without  hydrogen 
peroxide  a  FAINT  green  tinge  is  noticeable  only  after  40-50  min- 
utes. 

After  5  hours  4  did  not  show  a  trace  of  color.  1  and  3  again 
failed  to  exhibit  a  marked  contrast. 

Methods  of  visibly  demonstrating  the  absorption  of  oxygen 
were  now  attempted.  One  of  these  was  to  have  a  U-tube  ar- 
rangement attached  to  the  bottle  containing  thymol  and  am- 
monia. It  was  thought  that  as  the  oxygen  within  the  bottle 
would  be  absorbed,  the  difference  in  pressure  would  show  itself 
by  the  difference  in  the  level  of  liquid  in  the  U-tube ;  but  though 
the  color  appeared  no  difference  could  be  noticed.  Substituting 
a  narrower  U-tube  showed  alterations  in  level,  but  as  these  in- 
dicated expansion  at  one  time  and  contraction  at  another,  the 
cause  was  ascribed  to  differences  in  the  temperature  of  the 
room. 

Another  scheme  attempted  was  to  put  some  thymol  and 
ammonia  in  a  test  tube  and  invert  it  over  water,  the  mixture  but 
partly  filling  the  tube.  Any  oxygen  absorbed  would,  of  course, 
be  shown  by  a  rise  of  the  solution  in  the  tube.  But  here  again 
the  color  appeared  without  showing  any  increase  in  volume. 
However,  four  or  five  days  later,  when  the  solution  had  become 
much  darker  (more  intensely  blue)*,  the  volume  had  increased. 
Since  then,  several  repetitions  (in  all  cases  using  "controls"), 
have  confirmed  this  observation. 

The  addition  of  zinc  dust  to  the  solution  (thymol  0.5  gm. ; 
ammonium  hydroxid  solution  100  c.c.),  after  the  blue  color  had 
well  formed  (after  24  hours'  standing)  caused,  what  at  first  ap- 
peared to  be,  a  driving  of  the  color  to  the  surface,  leaving  the 
major  portion  of  the  solution  colorless.  Upon  closer  examination 
the  conclusion  was  reached  that  reduction  was  really  going  on,  that 
the  color  was  being  destroyed,  but  that  at  the  surface  there  was 
sufficient  oxygen  to  overcome  the  reducing  influence — a  suppo- 
sition which  the  shaking  of  the  solution  tended  to  confirm,  for 

*  The  color  formation  continues  for  days — even  weeks,  the  solution 
constantly  becoming  darker.  One  of  the  interesting  sequels  to  these 
observations  will  be  to  study  the  velocity  of  this  color  formation. 


44 

it  was  noticed  that  then  the  blue  color  spread  downward,  show- 
ing that  the  induced  air  was  overcoming  the  influence  of  the  hy- 
drogen evolved.  Ether  was  now  added — this  to  act  as  a  me- 
dium between  the  air  and  the  rest  of  the  solution.  The  bottle 
was  well  stoppered.  The  blue  that  was  in  the  solution  was 
extracted  by  the  ether,  forming  a  red  ether  layer.  The  next  morn- 
ing the  ether  layer  was  found  to  be  quite  colorless.  This  clearly 
suggested  that  the  nascent  hydrogen  had  succeeded  in  reducing 
the  colored  substance.  From  this  it  followed  that  by  loosening  the 
stopper  the  air  from  the  outside  would  once  again  recolor  the 
ether  layer.  And,  indeed,  within  five  minutes  the  ether  portion  be- 
came pink.  The  bottle  was  once  again  tightly  stoppered  and  al- 
lowed to  stand.  Three  hours  later  the  pink  color  had  altogether 
disappeared.  By  loosening  the  stopper  the  color  very  soon  re- 
appeared. 

These  experiments  were  repeated  many  times  on  the  same 
and  different  samples,  with  precisely  the  same  result.  We  are 
therefore  justified  in  concluding  that  nascent  hydrogen  destroys 
the  color,  the  oxygen  of  the  air  being  capable  of  reproducing  it. 

Attempts  were  now  made  to  isolate  this  reduced  compound. 
Having  in  mind  the  extreme  readiness  with  which  it  oxidizes, 
precautions  were  taken  to  prevent  this.  To  a  blue  solution  in 
a  flask  zinc  dust  and  ether  were  added.  Besides  the  hydrogen 
that  was  evolved  in  the  flask,  hydrogen  gas  was  passed  through 
the  entire  outfit  throughout  the  whole  of  the  experiment.  When 
the  ether  layer  had  become  colorless,  part  of  this  was  poured 
through  a  side  tube  into  a  perfectly  dry  flask,  which,  in  turn, 
was  connected  with  an  exhaust.  This  flask  was  now  surrounded 
with  warm  water,  the  evaporation  of  the  ether  being  hastened 
by  the  exhaust,  care  being  taken  to  so  regulate  this  that  no  air 
bubbles  were  introduced  into  the  hydrogen  generator.  But  the 
residue  that  was  left  was  reddish,  showing  that  it  had  oxidized 
in  some  way.  This  was  repeated  several  times,  the  utmost  pre- 
cautions being  taken  with  regard  to  leagage,  but  the  same  re- 
sults were  invariably  obtained. 

In  one  of  these  experiments  difficulties  were  experienced 
in  decolorizing  the  ether  layer.  It  was  recalled  that  the  blue 
solution  used  contained  alcohol,  and  it  was  thought  that  this 


45 

might  have  a  retarding-  influence.  With  the  object  of  testing 
this,  four  samples,  all  containing  the  blue  solution,  zinc  dust, 
and  ether,  were  taken,  and  to  two  of  them  alcohol  was  added. 
Even  on  the  addition  of  the  alcohol  a  blue  layer  was  formed 
between  the  pink  ether  layer  and  the  rest  of  the  solution  (a 
phenomenon  which  has  since  been  repeatedly  noticed.  Indeed, 
one  of  the  ways  of  hastening  the  oxidation  of  the  decolorized 
portion  is  to  add  alcohol):*  All  four  samples  were  allowed  to 
stand  overnight.  In  the  morning  it  was  found  that  the  ether 
layers  in  the  two  containing  alcohol  had  not  decolorized,  where- 
as the  other  two  had. 

In  another  experiment,  instead  of  starting  out  with  the  blue 
solution,  thymol,  zinc  dust,  ammonia,  and  ether  were  added  to 
the  flask.  This  was  allowed  to  stand  for  three  days,  but  no 
color  was  visible  in  the  ether  layer.  Part  of  the  latter  was 
now  poured  into  another  vessel,  and  the  ether  evaporated  (in 
a  manner  already  described).  What  remained  was  a  yellow 
liquid,  in  color  identical  with  that  obtained  by  the  action  of 
dry  ammonia  gas  upon  dry  thymol  after  it  had  been  allowed 
to  stand  for  a  little  while. 

In  several  other  experiments,  in  addition  to  the  zinc  dust, 
sodium  amalgam  was  also  added,  as  the  evolution  of  hydrogen 
was  far  more  rapid.  The  ether  layer  was  decolorized  in  about 
two  hours,  whereas  about  24  hours  was  required  without  the 
use  of  the  amalgam.  Here  purplish  particles  and  a  bluish  liquid 
constituted  the  residue  after  the  evaporation  of  the  ether. 

Several  other  arrangements  were  tried,  but  in  all  cases 
colored  deposits  were  left. 

At  this  point  a  study  was  made  of  the  action  of  dry  am- 
monia upon  dry  thymol.  Dry  ammonia  gas — prepared  by  heat- 
ing concentrated  ammonia  solution  and  passing  it  through  a 
soda  lime  tower — was  passed  into  thymol  from  which  all  moisture 
had  been  removed  by  keeping  it  in  a  drying  oven  for  one  to 
two  hours  around  50°,  and  leaving  it  in  an  exhaust  desiccator 

*  This  appears  contradictory  to  what  was  found  previously  (see  page 
32).  It  must  be  remembered,  however,  that  here  we  are  dealing  with  a 
substance  which  very  readily  takes  up  oxygen,  and  the  alcohol  may  be 
regarded  as  an  "oxygen  carrier." 


46 

overnight.  The  thymol  readily  absorbed  the  ammonia,  a  col- 
orless liquid  resulting.  This  solution,  however,  gradually  turned 
yellow,  then  brown,  and  finally  a  port  wine  color — all  in  the 
course  of  four  or  five  days. 

That  moisture  is  also  necessary  in  the  formation  of  color 
seems  to  be  indicated. 

EFFECT  OF  SEVERAL  REAGENTS  UPON  THE  BLUE  SOLUTION. 

A  closer  investigation  of  the  pigment  was  now  decided 
upon,  but  before  this  could  be  carried  out  a  convenient  and  rapid 
method  of  isolating  it  from  the  blue  solution  was  necessary. 

Gies  found  that  ether  extracts  the  blue  color  forming  a 
red  ether  layer  (1).  Chloroform  and  toluol  act  similarly.  Al- 
cohol, beyond  tending  to  emphasize  a  greenish  tinge,  seems  to 
have  no  marked  effect. 

Sulphuric  acid  converts  the  blue  color  into  red,  purplish 
drops  appearing  at  the  surface,  the  solution  being  very  cloudy. 
Hydrochloric  and  nitric  acids  act  similarly. 

When  the  red  ether  extract  is  evaporated,  an  amorphous 
purplish  mass  remains*  which  dissolves  in  sodium  hydroxide 
to  a  blue  solution,  and  this  in  turn  is  converted  into  a  red 
solution  by  sulphuric  acid — a  property  which  would  suggest  its 
use  as  an  indicator  (1). 

One  of  the  convenient  methods  of  extracting  the  blue  color 
was  to  dilute  the  solution  with  water,  neutralize  and  make 
slightly  acid  with  sulphuric  acid  (using  phenolphthalein  as  in- 
dicator), extract  with  ether,  and  evaporate  the  ether  portion.f 

*Subsequent  repetitions  gave  many  beautiful  blue  crystalline  deposits, 
but  further  work  led  to  the  belief  that  these  crystals  were  largely  com- 
posed of  unaltered  thymol — that,  in  fact,  they  were  thymol  crystals  col- 
ored by  the  pigment. 

t  This  method  required  much  less  ether.  The  same  end  could  be 
accomplished  by  nearly  saturating  the  blue  solution  with  salt  and  then 
extracting  with  ether.  For  fear  of  modifying  the  process  or  introduc- 
ing impurities,  neither  method  was  much  used,  direct  extraction  with 
ether  being  preferred. 


47 

TESTS  FOR  THE  PRESENCE  OF  NITROGEN   IN   THE  PIGMENT. 

The  opinion  had  been  formed  that  the  pigment  was 
thymoquinonethymolimid.  As  a  ready  means  of  testing  this  sup- 
position, it  was  decided  to  determine  the  amount  of  nitrogen 
in  the  substance. 

A  sample  of  the  blue  solution  was  extracted  with  ether  and 
the  ether  evaporated.  The  residue  was  dried  in  a  desiccator  for 
several  days,  and  two  portions  were  weighed  out:  A.  0.1165 
gm. ;  B.  0.1217  gm.  Each  was  treated  with  20  c.c.  of  sul- 
phuric acid  and  a  trace  of  copper  sulphate,  and  the  Kjeldahl 
run  in  the  ordinary  way.  The  ammonia  was  received  in  30  c.c. 
N/5  sulphuric  acid.  After  the  operation,  A  required  29.8  c.c. 
N/5  alkali  and  B  30  c.c. 

This  at  once  raised  certain  questions,  the  most  prominent 
of  which  was  whether  the  substance  was  really  the  genuine  blue  ?* 
It  was  quite  conceivable  that  the  presence  of  a  preponderating 
amount  of  some  other  substance,  nitrogen-free — possibly  un- 
changed thymol — would  account  for  this  result. 

Before  repeating  the  above  under  modified  conditions  a 
qualitative  test  for  nitrogen  was  made.  From  the  very  nature 
of  the  reaction  it  seemed  certain  that  the  blue  substance  con- 
tained nitrogen.  However,  the  quantitative  determination  gave 
rise  to  doubt. 

The  qualitative  test  was  made  by  fusing  a  sample  with 
sodium  and  treating  a  solution  of  the  product  with  ferric  and 
ferrous  salts  in  the  presence  of  hydrochloric  acid.  Not  a  trace  of 
Prussian  blue  was  obtained.  This  was  repeated  three  times  with 
the  same  result.  (Lassaigne's  test). 

In  the  possibility  that  some  substance  in  the  pigment  pre- 
vented the  reaction,  some  of  the  pigment  was  mixed  with  an- 
other substance  known  to  contain  nitrogen  (amidoacetophenone 
was  used),  but  here  no  difficulty  was  experienced  in  getting 
a  positive  result. 

These  results  seemed  most  perplexing.     It  appeared  in  the 

*  Thymoquinonethymolimid  should  have  yielded  about  4.5% 
nitrogen. 


highest  degree  improbable  that  the  pigment  contained  no  nitro- 
gen, and  yet  the  test  is  considered  a  reliable  one  even  for  com- 
parative traces  of  that  element.* 

Another  method  of  attacking  the  problem  was  now  consid- 
ered. If,  in  the  course  of  the  reaction  ammonia  is  taken  up, 
then  by  titrating  the  amount  that  remains,  the  necessary  data 
should  be  obtained. 

This  was  carried  out  by  dissolving  0.1  gm.  thymol  in  20 
c.c.  10%  ammonium  hydroxid  solution,  allowing  to  stand  for  two 
days,  diluting  to  200  c.c.,  extracting  the  blue  with  50  c.c.  ether, 
and  titrating  the  aqueous  portion,  25  c.c.  at  a  time.  Exactly  the 
same  treatment  was  accorded  a  "control,"  containing,  of  course, 
no  thymol.  (In  all  cases  the  ammonia  was  added  to  an  excess  of 
standard  acid,  and  the  excess  determined.)  Congo  red  served  as 
the  indicator.  Methyl  orange  could  be  substituted. 

Blue  Control 

25  c.c.  required  42.4  c.c.  acid  42.35  c.c. 

43.25      "  4245  c.c. 

43          "  42.25  c.c. 

43.1  "  42.35  c.c. 

200  c.c.  (20  c.c.  10%  ammonia)  342.5  338.7  c.c. 

This  actually  tended  to  show  that  there  was  more  ammonia 

*  Lassaigne,  the  originator  of  the  Prussian  blue  test  for  nitro- 
gen (45),  says:  "Le  precede  que  nous  avons  mis  en  pratique,  apres 
1'avoir  soumis  a  de  nombreux  essais,  est  si  sensible,  qu'il  permet  de 
reconnaitre  la  presence  de  1'azote  dans  des  quantites  de  matieres 
azotees  aussi  petites  que  celles  que  les  meilleures  balances  peuvent  a 
peine  apprecier." 

Graebe  (46)  failed  to  get  the  ferrocyanide  when  using  the  per- 
bromide  of  azonaphthalene.  Diazo  compounds  in  general  failed  to  give 
it,  as  the  nitrogen  was  evolved  at  a  low  temperature.  In  the  cases  of 
compounds  containing  sulphur,  unless  a  large  excess  of  sodium  was 
used,  the  test  would  not  always  be  successful. 

Jacobsen  (47)  cites  the  fact  that  the  sulphur  compound  produced  by 
the  oxidation  of  parasulphaminetoluic  acid  failed  to  give  the  nitrogen 
test,  though  by  using  an  excess  of  sodium  a  positive  result  was  ob- 
tained. 

Tauber  (48)  confirms  Graebe's  observations. 

See  Spiegel,  "Der  Stickstoff"  (1903),  p.  835.  Dr.  Heidelberger,  in 
a  private  communication  to  the  author,  stated  that  negative  results  were 
sometimes  obtained  by  the  use  of  too  large  a  quantity  of  ferric  chloride. 
The  merest  trace — just  sufficient  to  know  that  some  had  been  added — 
was  all  that  was  required. 


49 

at  the  end  of  the  reaction  than  at  the  beginning!    Several  repe- 
titions gave  equally  anomalous  results. 

Larger  quantities  of  thymol  were  now  used,  and  the  follow- 
ing mixtures  were  prepared : 

123456 

Thymol    2  gm.     5  gm.     10  gm.     15  gm.    20  gm.        — 

Ammonia    20  c.c.    20  c.c.     20  c.c.    20  c.c.    20  c.c.    20  c.c. 

These  samples  were  allowed  to  stand  for  two  days.  Into 
6  (counted  as  "control"),  5  gms.  of  thymol  were  added  just 
before  titration.  It  was  thought  that  possibly  the  presence 
of  thymol  would  prove  an  interfering  factor,  and  thereby  shed 
light  upon  the  results).  Each  was  made  up  to  100  c.c.  with 
water,  50  c.c.  ether  were  added,  shaken  well,  and  10  c.c.  with- 
drawn from  the  non-ether  layer.  This  was  added  to  50  c.c. 
N/5  acid  solution,  and  the  excess  titrated,  with  the  following 
results : 

1  required  38.9  c.c.  Average  for  20  c.c.  ammonia  387.75 

38.65  c.c. 

2  required  40.9  c.  Average  for  20  c.c.  ammonia  407.7 

40.65 

3  required  43.4  Average  for  20  c.c.  ammonia  433.5 

43.3 

4  required  44.35  Average  for  20  c.c.  ammonia  442.7 

44.2 

5  required  44.35  Average  for  20  c.c.  ammonia  442.7 

44.2 

6  required  39  Average  for  20  c.c.  ammonia  387.7 

38.55 

Here  a  similar  tendency  to  what  was  noticed  before  is 
shown ;  namely,  that  the  thymol,  instead  of  decreasing  the  amount 
of  ammonia,  increases  it!  But  these  results  can  be  explained 
satisfactorily.  Concentration  of  solutions  were  not  taken  into 
consideration.  Since  in  5  much  more  thymol  is  present  than  in 
1,  and  hence  takes  up  more  volume,  diluting  each  to  200  c.c., 
and  extracting  10  c.c.  from  each,  will  yield  a  more  concentrated 
solution  from  5  than  from  1. 

To  obviate  these  shortcomings  it  was  decided  to  titrate  the 
whole  rather  than  take  an  aliquot  portion. 

1  2  3 

Thymol    2  gm.  10  gm. 

Ammonia  (10%)    20  c.c.  20  c.c.  20  c.c. 


50 

(To  3  five  gms.  of  thymol  were  added  just  before  the  ti- 
tration.) 

The  solutions  were  allowed  to  stand  for  four  days.  20  c.c. 
ether  were  then  added  to  each  and  allowed  to  stand  for  another 
day.*  Each  was  then  poured  into  a  separating  funnel,  the  bot- 
tle rinsed  with  distilled  water,  and  the  washing  added  to  the  solu- 
tion in  the  sep.  funnel.  The  colorless  portion  (lower  layer)  was 
run  into  450  c.c.  sulphuric  acid,  and  the  residue  (the  red  ether 
layer),  washed  three  times  with  distilled  water,  and  the  wash- 
ings added.  To  test  the  residue  for  acid  it  was  again  washed 
with  water,  and  the  washings  run  into  a  known  volume  of  acid. 
This  was  repeated  until  all  the  acid  had  been  washed  out — as 
determined  by  titration. 

1  required  433.65   c.c.   acid. 

2  required  433.60  c.c.   acid. 

3  required  433.70  c.c.   acid.| 

These  results  were  the  most  trustworthy  as  every  possible 
precaution  was  taken.  Apparently,  these  tended  to  confirm  the 
indication  from  the  Kjeldahl  determination,  as  well  as  the  qualita- 
tive test,  that  the  pigment  did  not  contain  nitrogen. 

A  modified  Kjeldahl  for  nitro  compounds  (49)  was  now 
employed.  It  was  argued  that  possibly  the  nitrogen  was  in 
such  a  form  as  to  make  the  use  of  sulphuric  acid  alone  unre- 
liable. 

Two  samples  of  0.5  gm.  each  of  the  pigment  were  taken. 
To  each  30  c.c.  of  sulphuric  acid  containing  2  gms.  of  salicylic 
acid  were  added.  The  mixture  was  allowed  to  stand  for  twenty 
minutes.  After  the  addition  of  2  gms.  of  zinc  dust,  the  mix- 
ture was  first  gently  warmed,  and  then  heated  more  vigorously. 
Mercury  (about  1  gm.)  and  potassium  sulphate  (about  10  gms.) 
were  added,  and  the  whole  boiled.  The  boiling  was  continued 
for  about  1^  hours  after  the  solution  had  become  colorless. 

*It  is  needless  to  add  that  in  all  these  operations  perfect-fitting 
stoppers  for  the  bottles  were  used. 

f  These  figures  cannot  well  be  compared  with  the  foregoing  ones,  as 
the  ammonia  used  here  was  from  a  different  sample,  and  though  all 
the  samples  were  approximately  of  10%  strength,  none  of  them  was 
exactly  so. 


This  was  cooled  somewhat,  potassium  sulphide  and  excess  of  sod- 
ium hydroxide  were  added,  and  the  whole  distilled  into  75  c.c. 
of  N/o  acid  solution  : 

1  required    74.2    c.c.    alkali. 

2  required    74.5    c.c.    alkali. 
Control  required  74.1  c.c.  alkali. 

This  was  further  evidence  in  favor  of  what  had  been  found 
before. 

At  this  stage  attempts  were  begun  to  isolate  the  pure  pig- 
ment. As  is  recorded  elsewhere,  one  of  these  methods  was  to 
filter  a  blue  solution  that  had  been  standing  for  many  weeks. 
Naturally,  that  which  we  were  most  eager  to  do  was  to  test 
the  precipitate  on  the  filter  paper — a  sticky  mass — for  nitrogen. 
Some  of  this  filter  paper  was  cut  up,  well  soaked  in  ether,  and 
the  ether  solution  poured  into  two  tubes  in  which  the  nitrogen 
test  was  to  be  run.  These  were  warmed  to  50°  to  expel 
the  ether.  To  one  a  small  piece  of  sodium  was  added,  to  the 
other  potassium.  Neither  gave  an  immediate  test,  but  after 
standing  about  24  hours  a  distinct  Prussian  blue  precipitate  was 
detected  in  both  solutions.* 

ISOLATION  OF  THE  BLUE  PRODUCT. 

From  the  foregoing  work  it  became  clear  that  before  any 
further  headway  could  be  made,  a  method  of  isolating  the  pig- 
ment, uncontaminated  with  thymol  or  any  other  substance — 
one  that  may  be  formed  as  an  intermediate  product — would  have 
to  be  devised. 

For  some  time  in  the  past  it  had  been  recognized  that  the 
blue  crystals  must  consist  largely  of  unaltered  thymol — the  very 
shape  of  the  crystals  suggested  this.  Moreover,  the  fact  that 
they  gave  a  negative  nitrogen  test  simply  led  to  the  conclusion 
that  the  amount  of  colored  substance  present  was  very  small 
in  comparison  with  the  amount  of  thymol,  for  it  was  inconceiv- 
able from  the  very  nature  of  the  reaction  that  nitrogen  should 
be  absent.  The  task  now  was  to  find  a  complete  method  of 
separating  and  isolating  the  blue  product. 

*We  may  anticipate  here  and  state  that  in  subsequent  Kjeldahl  de- 
terminations where  the  pure  pigment  was  used,  nitrogen  was  invariably 
found  to  be  present. 


52 

1  gm.  of  thymol  and  150  c.c.  of  ammonium  hydroxid  solution 
were  placed  in  a  dialyzer  (parchment  paper)  and  suspended  in  wa- 
ter. After  seven  days  the  bag  was  opened,  and  the  solution  found 
to  be  perfectly  colorless,  though  parts  of  the  thymol  crystals 
showed  a  pink  color.  The  outer  solution  became  turbid  upon 
heating,  and  readily  responded  to  the  sulphuric — acetic  acid  test 
for  thymol.  A  blue  solution — that  is,  a  mixture  of  thymol  and 
ammonia  which  had  been  allowed  to  stand,  and  had  become 
blue — was  now  substituted  in  the  dialyzer,  and  suspended  in 
water.  After  some  time,  the  outer  solution  became  blue.  Evi- 
dently, then,  this  could  not  prove  a  method  of  separation. 

Bearing  in  mind  the  property  possessed  by  aluminum  hy- 
droxide of  carrying  down  coloring  matter  if  precipitated  in  the 
medium,  a  solution  of  aluminum  sulphate  was  added  to  the  blue 
liquid  (which,  of  course,  contained  ammonium  hydroxide). 
The  precipitate  was  allowed  to  stand  for  one  hour  and  fil- 
tered. A  flesh-colored  precipitate  was  obtained,  the  nitrate  show- 
ing just  the  slightest  bluish  tinge,  which,  however,  became  more 
pronounced  the  longer  it  stood.  When  the  precipitate  was  ex- 
tracted with  ether  only  a  part  went  into  solution.  By  evaporating 
the  ether  extract  a  violet  residue  was  left,  but  no  crystals  were 
evident. 

This  method  had  several  objections,  not  the  least  of  which 
was  the  time  element.  Attempts  were  now  made  to  prepare 
one  or  two  derivatives  of  thymol  in  the  hope  that  the  blue 
product  would  not  be  affected  thereby.  First,  the  formation  of 
thymol  iodide  was  tried.  Some  of  the  blue  crystals  were  dis- 
solved in  sodium  hydroxide,  and  to  the  solution  iodine  in  potas- 
sium iodide  was  added  till  a  precipitate  was  formed.  Upon  fil- 
tering the  filtrate  proved  to  be  colorless,  thus  showing  that  not 
only  the  thymol  but  the  blue  had  been  acted  upon. 

Next  the  benzoyl  derivative  was  attempted.  To  1  gm.  of 
the  crystalline  blue  0.5  gm.  water,  1  c.c.  benzoyl  chloride,  and 
sodium  hydroxide — till  the  solution  was  alkaline — were  added. 
The  mixture  was  stirred  till  the  smell  of  benzoyl  chloride  had 
practically  disappeared.  It  was  now  poured  into  a  large  ex- 
cess of  water.  The  expected  white  precipitate  was  obtained, 


53 

but  the  oily,  brownish  layer  on  top  showed  that  the  blue  had 
decomposed. 

It  was  noticed  that  in  all  the  blue  solutions  that  had  been 
standing  for  some  months  a  blue  sediment,  small  in  amount, 
had  settled  out.  A  sample  of  such  a  blue  solution  (which  we 
shall  label  X)  which  had  been  standing  for  about  3  months, 
was  filtered  (using  three  filter  papers).  The  filtrate  (Y)  was 
much  clearer  than  the  original  solution,  though  it  darkened  and 
became  opaque  on  standing.  On  the  filter  paper  there  was  a 
dark  purplish-bluish  mass,  oily  rather  than  solid  (Z).  From 
each  of  X  and  Y  some  solution  was  taken,  extracted  with  ether, 
and  the  ether  evaporated ;  Z  was  likewise  extracted  with  ether, 
and  the  solvent  evaporated.  The  ether  solution  of  X  was  dark 
violet  in  color;  of  Y  rose-red;  of  Z  violet  with  a  reddish  tinge. 
After  the  ether  had  been  evaporated,  X  showed  dark  violet 
crystals,*  Y  crystals  that  were  almost  colorless,  though  here  and 
there  tinged  with  a  light  violet,  and  Z  no  crystals  at  all,  but  a 
violet  sticky  mass  which  adhered  to  the  sides  of  the  beaker. 
Apparently  in  Z  no  thymol  was  present. 

This  method  of  separation  seemed  to  be  satisfactory  on 
the  face  of  it.  But  there  were  two  strong  objections  to  its  ex- 
tensive use:  a)  The  long  time  necessary  to  obtain  the  blue; 
b)  The  small  quantity  obtained.  To  obviate  the  first,  resort 
was  had  to  heating.  Quite  satisfactory  results,  similar  to  those 
recorded,  were  obtained.  Heating  on  the  water  bath  for  ten 
to  twelve  hours  accomplished  what  standing  at  room  tempera- 
ture would  have  taken  weeks  to  attain.  However,  too 
large  quantities  of  thymol  could  not  be  used,  as  the  thymol 
remained  suspended,  and,  of  course,  contaminated  the  precipi- 
tate. But  even  here  the  yield  of  blue  was  such  that  any  ex- 
tensive investigation  of  it  was  out  of  the  question. 

Another  method  was  now  tried.  Though  in  so  far  as  iso- 
lating the  blue  was  concerned  it  proved  a  failure,  yet  some  in- 
teresting results  were  obtained.  10  gms.  of  thymol  were  dis- 

*In  most  cases  where  these  crystals  would  form,  a  creeping  ten- 
dency on  their  part  would  be  exhibited.  Many  of  the  beakers  were 
found  to  be  covered  with  these  crystals  not  only  on  the  inside  but  out- 
side as  well. 


54 

solved  in  two  liters  of  ammonia  (IQ%).  The  next  day  30  gms. 
of  thymol  were  added,  and  immediately  part  of  it  turned  vio- 
let. Five  days  later  the  quantity  was  increased  with  20 
more  gms.  After  an  hour  or  so,  the  whole  solution  under- 
went a  complete  change.  From  blue  with  a  tinge  of  violet,  it 
now  became  violet  with  red  predominating.  A  deep  purple 
layer  formed  itself  on  top — as  if  ether  had  been  added  (1). 
Eleven  days  later  the  purple  layer  (A)  was  separated  from 
the  rest  (B).  To  B  twenty  more  gms.  of  thymol  were  added. 
It  was  reasoned  that  any  thymol  that  was  t  added  to  B  should 
cause  a  further  production  of  A,  since  B  already  had  a  large 
excess  of  thymol.  This  proved  to  be  the  case,  for  more  of  the 
purplish  variety  was  soon  obtained.  By  washing  A  several 
times  with  water  it  crystallized  out  to  a  violet  mass.  This 
crystallization  was  undoubtedly  due  to  the  presence  of  a  large 
quantity  of  unaltered  thymol. 

..&,       The  odor  of  A  was  most  peculiar.    It  recalled  thymol  some- 
:  .what,  though  quite  disagreeable.     Upon   covering  the  precipi- 
tate and  allowing  it  to  stand  for  several  days,  a  most  disagree- 
able odor,  vividly  suggesting  pyridine,  was  developed. 

Various  substances  were  now  tried  upon  thymol  and  the 
blue  pigment  in  the  hope  of  finding  a  medium  that  would  prove  a 
solvent  for  the  one  and  not  for  the  other  : 

1.  Water  Thymol  Pigment 

2.  Saturated   sodium  Insoluble  Insol. 
chloride  Insol.  Insol. 

3.  Alcohol  (95%)  Very  sol.  colorless  Very  sol.  Violet  (bluish) 

4.  Ether  V.  sol.  Colorless  sol.,  V.  sol.  Pink 

5.  Petrol,   ether  though  not  as  much  Sol.  Pink 

6.  Glacial    acetic   acid  V.  sol.  Colorless  V.  sol.  Violet  (reddish) 

7.  Benzine  V.  sol.  Colorless  V.  sol.  Pink 

8.  Carbon   bi-sulphide  V.  sol.  Colorless  V.  sol.  Violet  (bluish) 

9.  Acetone  V.  sol.  Colorless  V.  sol.  Violet  (bluish) 

10.  Chloroform  V.  sol.  Colorless  V.  sol.  Violet 

11.  Ethyl  acetate  V.  sol.  Colorless  V.  sol.  Violet 

12.  Methyl  alcohol  V.  sol.  Colorless  V.  sol.  Violet  (bluish) 

13.  Amyl  alcohol  Sol.  Colorless  Sol.  Violet  (bluish) 

14.  Toluene  V.  sol.  Colorless  V.  sol.  Violet  (reddish) 

15.  Xylene  V.  sol.  Colorless  V.  sol.  Violet  (reddish) 

16.  Lyridine  V.  sol.  Colorless  V.  sol.  Violet  (bluish) 

17.  Phenol  (water  solu-Insol.  (hot  and  cold)       Insol.  (hot  and  cold) 

tion)  Sol.  yellow  Sol.  Violet 

18.  Nitrobenzine  Sol.  yellow  Sol.  Violet  (bluish) 

19.  Aniline 


55 

The  common  acids  and  alkalis  proved  of  no  better  service. 

Concentrated  sulphuric  acid,  however,  forms  a  sulphonate 
which  is  soluble  in  water.  To  some  of  the  pigment,  then,  some 
cone,  sulphuric  acid  was  added,  and  the  solution  allowed  to 
stand  for  several  hours.  This  was  now  poured  into  a  large 
excess  of  water,  and  a  small  quantity  of  a  greenish-colored  pre- 
cipitate was  obtained. 

As  thymol  is  volatile  with  steam,  this  was  thought  of  as 
a  possible  means  of  separation,  and  it  proved  very  good.  When 
the  pigment  was  steam  distilled,  thymol  came  over,  leaving  the 
pure  pigment  behind.  The  steam  distillation  was  continued  till 
the  distillate  no  longer  gave  a  sulphuric  acid — glacial  acetic  acid 
test  (a  "control"  was  used,  for  even  with  a  "control"  a  slight 
pink  is  obtained).  The  solution  in  the  distilling  flask  was  now 
extracted  with  ether  (sodium  chloride  was  for  some  time  used 
to  first  saturate  the  solution,  but  this  contaminated  the  ether 
extract,  and  was  eventually  discarded),  and  the  ether  evaporated. 
An  intense  deep  purple  product  was  obtained,  but  not  a  trace 
of  crystalline  matter. 

Though  this  method  does  not  yield  the  substance  in  a  crys- 
talline form,  as  does  the  sulphuric  acid  method,  yet  in  subse- 
quent work  it  was  preferred  because :  a)  Purity  of  product 
was  more  assured;  b)  product  more  easily  obtained  and  in 
larger  quantities ;  and  c)  less  time  required. 

THE  CHEMICAL  CONSTITUTION   OF  THE  PIGMENT. 

The  work  done  so  far  does  not  justify  definite  conclusions 
with  regard  to  the  exact  chemical  nature  of  the  compound  (or 
compounds)  for  it  is  probably  a  mixture  obtained  by  the  ac- 
tion of  ammonia  or  thymol.  However,  clues  to  the  path  to 
be  traced  are  not  wanting. 

Wurster  (36),  in  the  article  so  frequently  referred  to,  states 
that  his  blue,  obtained  by  the  action  of  hydrogen  peroxide  and 
ammonia  on  phenol,  is  phenolquinoneimid.  This  he  prepared 
in  two  other  ways :  1)  By  the  addition  of  ammonia  to  a  watery 
solution  of  quinone  in  excess  of  phenol ;  2)  by  dissolving  />-amido- 
phenol  in  sodium  hydroxide  (which  becomes  yellow  in  contact 
with  air)  and  adding  phenol.  Hirsch  (37)  prepared  the  same 


56 

phenolquinoneimid  by  dissolving  quinonechlorimid  in  phenol  in 
the  presence  of  sodium  hydroxide.  Wurster  carefully  differen- 
tiates his  pigment  from  that  obtained  by  Liebermann  (50),  differ- 
ences in  spectroscopic  behavior  and  in  the  colors  obtained  by 
the  addition  of  ether  to  acid  solutions,  justifying  his  conclusion.* 
Thymol,  he  records,  behaves  similarly. 

Now,  if  this  be  correct,  then  Decker  and  Solonina's  view 
that  the  Liebermann  pigment  obtained  from  thymol  (prepared 
by  the  action  of  nitrous  acid  on  thymol)  is  also  thymoquinone- 
thymolimid  (51)  is  erroneous. 

From  our  own  observations  so  far.  we  have  been  led  to 
believe  that  the  two  are  different. 

Some  of  Liebermann's  pigment  was  prepared  and  compared 
with  our  own,  with  the  results  indicated  below : 

Liebermann's  Our  pigment 

Water  Insol.  reddish  Insol.   violet 

Alcohol  Purple    (red)  Purple  (blue) 

Ether  Red  Violet    (various   shades) 

Glacial  acetic  Orange-red  Violet 

Amyl  alcohol  Purple   (red)  Violet  (blue) 

Liebermann's  pigment,  when  steam  distilled,  taken  up  with 
ether,  and  the  ether  evaporated,  gives  the  same  pasty  residue  as 
ours,  but  the  color  is  much  redder.  Whereas  our  pigment,  when 
dissolved  in  cone,  sulphuric  acid,  and  the  solution  poured  into 
water  gives  a  very  fine  greenish-blue  precipitate,  which  is  almost 
impossible  to  filter,  Liebermann's  gives  a  flocculcnt  reddish  pre- 
cipitate, readily  filtered. 

That  the  blue  pigment  may  prove  to  be  a  mixture  of  more 
than  one  substance — one  of  which  is  probably  thymoquinone- 
thymolimid — does  not  seem  at  all  improbable,  for  various 
shades  of  the  colored  pigment  (the  violet  varying  from  the  red  to 
blue)  have  been  obtained. 

*"Da  die  saure  Losung  des  Farbstoffs  in  Aether  roth  ist,  wahrend 
der  Liebermannsche  Farbstoff  aus  verdiinnter  saurer  Losung  in  Aether 
mit  gelber  Farbe  ubergeht,  so  ist  die  Verschiedenheit  der  beiden  Kor- 
per  schon  wahrscheinlich ;  dies  geht  auch  aus  dem  spectroskopischen 
verhalten  des  Farbstoffs  hervor,  welcher  zwar  eine  nicht  scharf 
begrenzte  Absorption  im  Roth  zeigt  aber  nicht  den  charakteristischen 
Streifen  der  Liebermann'schen  Farbstoff." 


57 

THE  ACTION  OF  SODIUM  HYDROXIDE,  POTASSIUM 

HYDROXIDE  AND  BARIUM   HYDROXIDE  ON 

THYMOL. 

Early  in  these  investigations  it  became  of  interest  to  com- 
pare the  action  of  ammonia  with  that  of  other  alkalis,  with  the 
view  to  ascertaining  whether  the  blue  color  formation  was  due 
specifically  to  ammonia  or  merely  because  ammonia  belonged  to 
the  class  of  alkalis.  In  this  respect  very  few  experiments  were 
needed  to  show  how  specific  in  function  was  the  ammonia. 

Sodium  hydroxide  developed  a  pink  color  (Table  XVIII), 
the  intensity  of  which  decreased  with  increasing  quantities  of 
alkali  (Table  XIX),  though  it  increased  up  to  a  certain  limit 
(Table  XX),  and  increased  in  the  presence  of  alcohol  (Tables 
XXI  and  XXII).  Heat  accelerates  the  color  formation  (Table 
XXIII).  The  red  color  could  not  be  extracted  with  ether,  but 
upon  evaporating  the  ether  portion,  thymol  was  recovered  (Note 
A).  The  action  of  zinc  dust  was  somewhat  peculiar  (Note  B). 

Potassium  hydroxide  acted  similarly  (Tables  XXV  and 
XXVI).  Barium  hydroxide  failed  to  give  any  color — possibly 
owing  to  the  weakness  of  the  solution  (Table  XXVII). 

TABLE  XVIII. 

Comparative  action  of  sodium  hydroxide  and  ammonium 
hydroxide  upon  thymol,  alcohol  being  absent. 

1.  2. 

Thymol    0.5  gm.    Thymol    0.5  gm. 

Sodium  hydroxide  (10%)*      10  c.c.    Ammonia  (10%)    100  c.c. 

Water    100  c.c.    Water    10  c.c. 

Conclusion. — Whereas  2  develops  the  usual  green  to  green- 
ish-blue color,  1  develops  a  pink. 

Notes. — It  took  from  24-36  hrs.  to  develop  a  pinkish  tinge 
in  1. 

Used  more  of  ammonia  than  sodium  hydroxide  as  latter  is 
a  stronger  base. 

*Unless  otherwise  stated,  the  sodium  hydroxide  used  throughout  was 
10%. 


TABLE  XIX. 

Effect  of  different  quantities  of  sodium  hydroxid  upon  a 
constant  quantity  of  thymol. 

12345 

Thymol  0.5  gm.    0.5  gm.    0.5  gm.    0.5  gm.    0.5  gm. 

Sodium  hydroxide   10  c.c.      20  c.c.      30  c.c.      40  c.c.      50  c.c. 

Water   50  c.c.      40  c.c.      30  c.c.      20  c.c.      10  c.c. 

Conclusion. — Pinkish  color  develops,  most  pronounced  in 
1,  least  in  5.  That  is,  intensity  of  color  decreases  with  in- 
creasing quantities  of  sodium  hydroxide. 

Notes. — Within  two  hours  a  pink  tint  can  be  noticed  in 
all. 

The  intensity  of  color  seems  to  reach  a  maximum  in  about 
48  hours  and  then  does  not  seem  to  change  (whereas  when  am- 
monia is  used  the  color  deepens  continuously). 

Two  days  after  the  maximum  color  had  been  reached,  the 
pink  color  had  disappeared  altogether.  10  c.c.  of  alcohol  were 
then  added  to  each.  A  pinkish  color  now  gradually  developed, 
being  most  intense  in  1,  least  in  5. 

TABLE  XX. 

What  amount  of  sodium  hydroxid,  keeping  thymol  and  al- 
cohol constant,  would  give  the  maximum  color? 

12345 

Thymol  0.5  gm.    0.5  gm.    05  gm.    0.5  gm.    0.5  gm. 

Sodium  hydroxide    2  c.c.        4  c.c.        6  c.c.        8  c.c.       10  c.c. 

Water   58  c.c.      56  c.c.      54  c.c.      52  c.c.      50  c.c. 

Alcohol  10  c.c.      10  c.c.      10  c.c.      10  c.c.      10  c.c. 

Conclusion. — Maximum  coloration  obtained  with  10  c.c. 
sodium  hydroxide  solution  (10%). 

Notes. — Color  least  intense  in  1,  most  in  5;  and  from  pre- 
vious work  it  was  found  that  when  more  than  10  c.e.  sodium 
hydroxide  solution  is  present,  the  color  is  less  intense. 


59 
TABLE  XXI. 

Effect  of  variable  quantities  of  sodium  hydroxid  upon  a  con- 
stant quantity  of  thymol,  in  the  presence  of  a  constant  quantity  of 
alcohol. 


Thymol  

1 
05  gm 

2 
0.5  gm. 

3 
0.5  gm. 

4 

0  S    crm 

5 

O  S    crm 

Sodium  hydroxide  
Water  
Alcohol  .. 

.     10  c.c. 
.     50  c.c. 
10  c.c. 

20  c.c. 
40  c.c. 
10  c.c. 

30  c.c. 
30  c.c. 
10  c.c. 

u.o  gm. 
40  c.c. 
20  c.c. 
10  c.c. 

u.o  grn. 
50c.c. 
10  c.c. 
10  c.c. 

Conclusion. — Intensity  of  color  varies  inversely  with 
amount  of  sodium  hydroxide  present.  Color  increased  by  pres- 
ence of  alcohol. 

Notes. — Though  this  series  was  prepared  27  hours  later  than 
the  one  containing  no  alcohol,  the  colors  were  far  more  intense ; 
so  much  so,  that  the  least  intense  here  was  found  to  be  more 
colored  than  the  most  intense  there. 

TABLE  XXII. 

Effect  of  different  quantities  of  alcohol  upon  a  relatively 
large  quantity  of  thymol  in  the  presence  of  a  constant  quantity 
of  sodium  hydroxide. 

12345 

Thymol  1  gm.       1  gm.        1  gm.       1  gm.       1  gm. 

Sodium  hydroxide   10  c.c.      10  c.c.      10  c.c.      10  c.c.      10  c.c. 

Alcohol  10  c.c.      20  c.c.      30  c.c.      40  c.c.      50  c.c. 

Water  50  c.c.      40  c.c.      30  c.c.      20  c.c.      10  c.c. 

Conclusion. — Color  goes  from  pink  to  brown  in  proceeding 
from  1-5. 

TABLE  XXIII. 

Effect  of  heat  upon  the  development  of  the  pink  color  by 
the  action  of  sodium  hydroxide  upon  thymol  in  the  presence  of 
alcohol. 

Thymol 0.5  gms. 

Sodium  hydroxide   50  c.c. 

Alcohol  10  c.c. 

Conclusion. — Color  developed  in  15-20  minutes. 


6o 
NOTE  A. 

Action  of  ether  upon  the  red  solution  formed  by  the  action  of 
sodium  hydroxide  upon  thymol  in  the  presence  of  alcohol. 

To  a  small  quantity  of  the  red  solution  (taken  from  a  bot- 
tle in  which  the  red  solution  had  been  kept  for  days,  and  which 
was  getting  darker  and  more  brownish  with  time),  some  water 
was  added,  shaken,  and  then  some  ether  added  (the  object  of 
the  water  was  to  dissolve  out  the  alcohol,  and  thereby  lessen 
its  effect  upon  the  ether).  It  was  hoped  that  the  color  (as  in 
the  case  of  the  NH4OH  thymol  blue  formation)  would  be  ex- 
tracted by  the  ether,  but  no  such  thing  happened.  On  allow- 
ing to  stand  overnight  it  was  found  that  the  color  of  the  solu- 
tion had  changed  from  a  dark  brownish-red  to  a  light  yellow, 
the  ether  layer  on  top  remaining  colorless.  For  4  days  no  fur- 
ther change  was  visible.  By  separating  the  ether  layer  from  the 
rest  of  the  solution,  and  evaporating  the  ether  rhombic  crys- 
tals, resembling  thymol  when  a  concentrated  solution  in  acetic 
is  poured  into  water,  were  obtained.  The  m.  p.  of  this  (48°) 
strengthens  this  belief. 

NOTE  B. 

Action  of  Zn  dust  upon  the  red  solution  formed  by  the  action 
of  sodium  hydroxide  upon  thymol  in  the  presence  of  alcohol. 

To  a  red  solution  composed  of  thymol  0.5  gms.,  sodium 
hydroxide  solution  (10%)  10  c.c.,  water  50  c.c.,  alcohol  10  c.c., 
and  which  had  been  standing  almost  a  month,  some  Zn  dust  was 
added.  In  15-20  minutes  the  solution  had  become  completely  de- 
colorized. But  the  pink  again  appeared — this  time  on  top.  The  ad- 
dition of  ether  caused  the  pink  to  be  taken  up  by  the  ether  as  pink. 

TABLE  XXIV. 

Effect  of  various  mixtures  of  sodium  hydroxide  and  am- 
monia upon  a  constant  quantity  of  thymol. 

123456789 

Thymol     0.5  gm.  .5  gm.  .5  gm.  .5  gm.  .5  gm.  .5  gm.  .5  gm.  .5  gm.  .5  gm. 

Ammonia    90  c.c.  80  c.c.  70  c.c.  60  c.c.  50  c.c.  40  c.c.  30  c.c.  20  c.c.  10  c.c. 

Sodium  hydroxide  10  c.c.  20  c.c.  30  c.c.  40  c.c.  50  c.c.  60  c.c.  70  c.c.  80  c.c.  90  c.c. 


6i 

TABLE  XXIV. — Continued. 

After  45  hours— 1  ,  Greenish-blue ;  2,  less  intense ;  3,  faintest 
greenish  tinge;  4,  faint  pink;  5,  somewhat  more  so;  6,  about 
same;  7,  lighter;  8,  still  lighter;  9,  almost  no  pink. 

Two  days  later. — 1,  A  sort  of  dark,  somewhat  dirty  green, 
radically  differing  from  ammonia,  thymol  alone  (which  becomes 
blue)  ;  2,  same,  but  much  lighter;  3,  slight  pink  with  a  shade  of 
the  green;  4,  light  pink;  5,  6,  slightly  more  intense;  7,  slightly 
less ;  9,  less,  just  faint  pink. 

TABLE  XXV. 

Effect  of  variable  quantities  of  potassium  hydroxide  upon 
a  constant  quantity  of  thymol,  in  the  presence  of  a  constant 
quantity  of  alcohol. 

12345 

Thymol     0.5  gtn.      0.5  gm.      0.5  gm.      0.5  gtn.      0.5  gm. 

*Potassium    hydroxide..     10  c.c.        20  c.c.        30  c.c.        40  c.c.        50  c.c. 

Water    50  c.c.        40  c.c.        30  c.c.        20  c.c.        10  c.c. 

Alcohol   (95%)    10  c.c.        10  c.c.        10  c.c.        10  c.c        10  c.c. 

Conclusion. — Pink  color,  gradually  tending  to  brown  ob- 
tained; most  in  1,  least  in  5. 

Notes. — Differences  in  intensity  of  the  pink,  when  first  de- 
veloped, could  not  be  distinguished. 

Five  days  after  commencing  the  experiment  the  pink  had 
almost  wholly  given  way  to  brown,  the  intensity  remaining  as  be- 
fore. 

TABLE  XXVI. 

Effect  of  different  quantities  of  potassium  hydroxide  upon 
a  constant  quantity  of  thymol  in  the  absence  of  alcohol. 

12345 

Thymol    0.5  gm.      0.5  gm.      0.5  gm.      0.5  gm.      0.5  gm. 

Potassium   hydroxide....     10  c.c.        20  c.c.        30  c.c.        40  c.c.        50  c.c. 
Water    50  c.c.        40  c.c.        30  c.c.        20  ac.        10  c.c. 

Conclusion. — Pink  color  developed,  most  in  1,  least  in  5. 

Notes. — After  40  hours'  standing,  there  wae  but  the  faintest 
trace  of  pink  visible.     Within  the  next  24-30  hours  it  had  in- 
creased appreciably,  though  the  color  still  remained  very  slight. 
*  Unless  otherwise  stated  the  strength  of  the  potassium  hydroxide 
used  was  10%. 


62 

After  5  days  the  color  had  disappeared,  and  had  given  place 
to  a  slight  brownish  color;  most  in  1,  least  in  5. 

On  the  7th  day,  10  c.c.  alcohol  added  to  each.  A  pink  color 
gradually  developed  in  the  next  12  hours.  At  first  the  intensity 
was  about  the  same  in  all,  but  2  days  later  it  was  most  marked 
ir:  1,  least  in  5. 

TABLE  XXVII. 

Effect  of  different  quantities  of  barium  hydroxide  upon  a 
constant  quantity  of  thymol  in  the  presence  of  a  constant  amount 
of  alcohol. 

12345 

Thymol   0.5  gm.    0.5  gm.    0.5  gm.    0.5  gm.    0.5  gin. 

Barium  hydroxide  (sat.  sol.)     10  c.c.      20  c.c.      30  c.c.      40  c.c.      50  c.c. 

Water   50  c.c.      40  c.c.      30  c.c.      20  c.c.      10  c.c. 

Alcohol  10  c,c.      10  c.c.      10  c.c.      10  c.c.      10  c.c. 

Conclusion. — No  color. 

Notes. — Though  standing  for  17  days,  no  color  developed. 
All  cloudy.  1  showed  a  beautiful  hexagonal  crystal,  slightly 
pinkish  around  its  borders ;  2  showed  several  such  crystals  of 
smaller  size,  joined  together,  with  pink  more  pronounced ;  3  and 
4  had  circular  oily  layer  on  top,  of  same  color  as  solution  (cloudy- 
white)  ;  5  same,  but  slightly  pinkish. 

The  crystal  in  (1)  was  taken  out.  When  broken  up,  it 
was  found  to  smell  strongly  of  alcohol.  It  would  seem  as  if 
the  alcohol  had  concentrated  around  the  point.  The  crystal, 
though  pinkish,  when  finely  ground,  gave  a  fine  white  powder, 
resembling  thymol.  Its  m.  p.  (49°)  strengthens  the  assump- 
tion. 

ADDENDUM. 

In  the  course  of  one  of  the  experiments  it  was  noticed  that 
by  additions  of  a  fairly  large  excess  of  thymol  to  a  10%  am- 
monium hydroxide  solution  in  a  glass-stoppered  bottle,  the  blue 
color  tended  gradually  to  disappear.  In  time  a  completely  color- 
less, though  slightly  turbid,  solution  was  obtained,  with  a  purplish 
oily  layer  on  top.  Not  the  least  remarkable  was  the  behaviour  of 


63 

this  solution  with  temperature  changes.  Warming  around  30° 
caused  the  solution  to  become  quite  opaque;  cooling  to  about 
17°C  again  gave  a  clear  greenish  solution.  Gradually  a  pale 
green  to  blue  crystalline  substance  separated  out,  and  fell  to  the 
bottom. 

When  the  bottle  was  opened  and  shaken  up  with  a  new  sup- 
ply of  air,  the  colorless  solution  became  immediately  deeply 
bluish.  This  implies  that  the  substance  was  reduced. 

SUMMARY  OF  GENERAL  CONCLUSIONS. 

1.  The  melting  point  of   thymol,   variously  given   in   the 
literature  from  44°  to  53°,  is  found  to  be  50-50.5°. 

2.  The  blue  color  obtained  by  the  action  of  ammonia  upon 
thymol  is  not  due  to  any  impurity  in  the  preparations  of  the 
latter. 

3.  Within   certain   limits   the  intensity  of  the  blue  color 
varies  directly  with  the  amount  of  thymol  present. 

4.  Within   certain   limits   the   intensity   of   the  blue  color 
varies  directly  with  the  amount  of  ammonia  present. 

5.  Within  certain  limits  alcohol  does  not  effect  the  blue 
color  formation. 

6.  One  part  of  thymol  in  25,000  parts  of  10%  ammonium 
hydroxide  solution  shows  a  distinct  color  within  five  days. 

7.  Heat  and  hydrogen  peroxide  accelerate  the  production 
of  the  pigment;  cold  and  hydrogen  have  a  retarding  effect;  light 
has  no  influence;  ether  inhibits  the  color  formation. 

8.  Oxygen  is  an  essential  factor  in  the  color  formation. 

9.  Moisture  is  probably  also  necessary. 

10.  Nascent  hydrogen  destroys  the  red  color  obtained  by 
extracting  the  blue  solution  with  ether,  the  oxygen  of  the  air 
being  capable  of  reproducing  it. 

11.  Dry  thymol  absorbs  dry  ammonia  gas  forming  a  col- 
orless liquid. 

12.  The  pigment  isolated  by  extracting  the  blue  solution 
with  ether  and  evaporating  the  latter  fails  to  show  the  pres- 
ence of  nitrogen.     This  negative  result  is  due  to  the  presence 
of  preponderating  amounts  of  unaltered  thymol   (and  possibly 
also  intermediate  products?). 


64 

13.  The  most  convenient  method  of  isolating  the  blue  pig- 
ment in  the  pure  state  is  to  submit  the  crude  product  as  ob- 
tained in  12  to  steam  distillation  till  the  distillate  fails  to  re- 
spond to  thymol  tests. 

14.  The  belief  is  expressed  that  the  pigment  is  probably 
a  mixture  of  two  or  more  substances,  one  of  which  may  pos- 
sibly be  thymoquinonethymolimid. 

15.  The  pigment  possesses  properties  that  point  to  its  value 
as  an  indicator. 

16.  The  action  of  sodium  hydroxide  upon  thymol   is   to 
produce  a  pink  color,  the  intensity  of  which  is  increased  by  al- 
cohol.   Increased  quantities  of  sodium  hydroxide  do  not  increase 
the  intensity  of  the  color.     The  color  can  not  be  extracted  with 
ether  from  the  solution. 

17.  Potassium  hydroxide  acts  similarly  to  sodium  hydrox- 
ide. 

18.  Barium  hydroxide  does  not  produce  any  color. 


65 
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BIOGRAPHICAL. 

Benjamin  Horowitz  was  born  on  August  25,  1888.  He  re- 
ceived his  secondary  school  and  college  training  at  Raines 
Foundation  School,  London,  Eng.,  and  Finsbury  College,  Lon- 
don, Eng.  In  the  fall  of  1907  he  entered  the  School  of  Chemis- 
try, School  of  Applied  Science,  Columbia  University,  and  in 
1911  he  received  the  degree  of  Chemist.  During  1911-1912  he 
pursued  post-graduate  work  in  Organic  Chemistry,  and  in  1912 
he  received  the  degree  of  Master  of  Arts.  During  the  past  year 
he  has  been  engaged  in  research  work  in  the  department  of 
Biological  Chemistry,  Columbia  University. 

In  the  fall  of  1911  he  was  appointed  assistant  in  Organic 
Chemistry,  Clark  University,  Worcester,  Mass.,  and  in  February, 
1913,  he  received  a  similar  appointment  in  the  Department  of 
Biological  Chemistry,  Columbia  University. 


68 


PUBLICATION. 

Experiments  on  pigments  produced  from  thymol  by  the  ac- 
tion of  ammonia.    Biochem  Bulletin,  2,  293  (1913). 


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